Hepatitis C subtype distribution in chronically infected patients with mild liver fibrosis in France: the GEMHEP study.

Treatment options for Hepatitis C infection have greatly improved with direct-acting antiviral (DAA) combinations achieving high cure rates. Nevertheless, the cost of this treatment is still high and access to treatment in many countries has been preferentially reserved for patients with more severe fibrosis (F3 and F4). In this French nationwide study, we investigated the epidemiological characteristics and genotype distribution of hepatitis C virus (HCV) in treatment-naive patients with METAVIR fibrosis stages between F0 and F2 in order to identify patient profiles that became eligible for unrestricted treatment in a second period. Between 2015 and 2016 we collected data from nine French university hospitals on a total of 584 HCV positive patients with absent, mild or moderate liver fibrosis. The most represented genotypes were genotype 1b (159/584; 27.2%), followed by genotype 1a (150/584; 25.7%); genotype 3 (87/584: 14.9%); genotype 4 (80/584; 13.7%). Among genotype 4: 4a was predominantly encountered with 22 patients (27.5% of genotype 4). Genotypes 1b and 1a are currently the most frequent virus types present in treatment-naive patients with mild fibrosis in France. They can be readily cured using the available DAA. Nevertheless, non-a/non-d genotype 4 is also frequent in this population and clinical data on the efficacy of DAA on these subtypes is missing. The GEMHEP is the French group for study and evaluation of viral hepatitis on a national scale. Data collection on epidemiological and molecular aspects of viral hepatitis is performed on a regular basis in all main French teaching hospitals and serves as a basis for surveillance of these infections. Analysis and trends are regularly published on behalf of the GEMHEP group. Data collection was performed retrospectively over the 2015-2016 period, covering nine main university hospitals in France. A total of 584 hepatitis C positive patients were included in this study. Genotyping of the circulating viruses showed a high prevalence of genotypes 1b and 1a in our population. The epidemiology of hepatitis C is slowly changing in France, particularly as a consequence of the rise of 'non-a non-d' genotype 4 viruses mainly originating from African populations. More data concerning treatment efficacy of these genotypes is needed in order to guide clinical care.

Emerging resistance mutations in PI-naive patients failing an atazanavir-based regimen (ANRS multicentre observational study).

Background: Atazanavir is a PI widely used as a third agent in combination ART. We aimed to determine the prevalence and the patterns of resistance in PI-naive patients failing on an atazanavir-based regimen. Methods: We analysed patients failing on an atazanavir-containing regimen used as a first line of PI therapy. We compared the sequences of reverse transcriptase and protease before the introduction of atazanavir and at failure [two consecutive viral loads (VLs) >50 copies/mL]. Resistance was defined according to the 2014 Agence Nationale de Recherche sur le SIDA et les Hépatites Virales (ANRS) algorithm. Results: Among the 113 patients, atazanavir was used in the first regimen in 71 (62.8%) patients and in the first line of a PI-based regimen in 42 (37.2%). Atazanavir was boosted with ritonavir in 95 (84.1%) patients and combined with tenofovir/emtricitabine or lamivudine (n = 81) and abacavir/lamivudine or emtricitabine (n = 22). At failure, median VL was 3.05 log10 copies/mL and the median CD4+ T cell count was 436 cells/mm3. The median time on atazanavir was 21.2 months. At failure, viruses were considered resistant to atazanavir in four patients (3.5%) with the selection of the following major atazanavir-associated mutations: I50L (n = 1), I84V (n = 2) and N88S (n = 1). Other emergent PI mutations were L10V, G16E, K20I/R, L33F, M36I/L, M46I/L, G48V, F53L, I54L, D60E, I62V, A71T/V, V82I/T, L90M and I93L/M. Emergent NRTI substitutions were detected in 21 patients: M41L (n = 2), D67N (n = 3), K70R (n = 1), L74I/V (n = 3), M184V/I (n = 16), L210W (n = 1), T215Y/F (n = 3) and K219Q/E (n = 2). Conclusions: Resistance to atazanavir is rare in patients failing the first line of an atazanavir-based regimen according to the ANRS. Emergent NRTI resistance-associated mutations were reported in 18% of patients.

Quality control implementation for universal characterization of DNA and RNA viruses in clinical respiratory samples using single metagenomic next-generation sequencing workflow.

BACKGROUND: In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on clinical respiratory samples and implementation of quality controls (QCs) is still lacking. METHODS: A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7). RESULTS: The optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R2 ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses). CONCLUSIONS: Although the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.

Prevalence of HIV-1 drug resistance in treated patients with viral load >50 copies/mL: a 2014 French nationwide study.

Background: Surveillance of HIV-1 resistance in treated patients with a detectable viral load (VL) is important to monitor, in order to assess the risk of spread of resistant viruses and to determine the proportion of patients who need new antiretroviral drugs with minimal cross-resistance. Methods: The HIV-1 protease and reverse transcriptase (RT) and integrase genes were sequenced in plasma samples from 782 consecutive patients on failing antiretroviral regimens, seen in 37 specialized centres in 2014. The genotyping results were interpreted using the ANRS v24 algorithm. Prevalence rates were compared with those obtained during a similar survey conducted in 2009. Results: The protease and RT sequences were obtained in 566 patients, and the integrase sequence in 382 patients. Sequencing was successful in 60%, 78%, 78% and 87% of patients with VLs of 51-200, 201-500, 501-1000 and >1000 copies/mL, respectively. Resistance to at least one antiretroviral drug was detected in 56.3% of samples. Respectively, 3.9%, 8.7%, 1.5% and 3.4% of patients harboured viruses that were resistant to any NRTI, NNRTI, PI and integrase inhibitor (INI). Resistance rates were lower in 2014 than in 2009. Resistance was detected in 48.5% of samples from patients with a VL between 51 and 200 copies/mL. Conclusion: In France in 2014, 90.0% of patients in AIDS care centres were receiving antiretroviral drugs and 12.0% of them had VLs >50 copies/mL. Therefore, this study suggests that 6.7% of treated patients in France might transmit resistant strains. Resistance testing may be warranted in all treated patients with VL > 50 copies/mL.

Resistance training as supportive measure in advanced cancer patients undergoing TKI therapy-a controlled feasibility trial.

PURPOSE: While there is growing evidence for positive effects of progressive resistance training in curatively treated cancer patients, data on advanced cancer patients are scarce. This pilot study aimed at investigating for the first time feasibility and effects of progressive resistance training in advanced cancer patients undergoing tyrosine kinase inhibitor (TKI) therapy. METHODS: Patients starting a TKI-based anti-tumor therapy were assigned to a resistance training group (RT, 12 weeks of progressive machine-based resistance training 2×/week) or a control group (CON, treatment as usual) until 10 patients had finished in each group (RT 80% males, 90% renal cell carcinoma, 65 ± 11 years, CON 80% males, 70% renal cell carcinoma, 61 ± 6 years). Primary endpoint was feasibility. Furthermore, fatigue (MFI), quality of life (QoL, EORTC QLQC30), and muscle strength were assessed. Testing occurred at baseline and after 12 weeks. RESULTS: Training was feasible in 9 out of 10 participants and no serious adverse events occurred. It had beneficial effects on muscle strength (maximum voluntary isometric contraction of the quadriceps: RT +11 ± 9 Nm, CON -13 ± 25 Nm, p = 0.005), but not on fatigue (general fatigue score RT +0.3 ± 4.1, CON -1.5 ± 3.0, p = 0.223) or QoL (global QoL score RT -5.6 ± 16.1, CON -2.0 ± 18.2, p = 0.617). CONCLUSIONS: Progressive machine-based resistance training appears feasible in the majority of advanced cancer patients undergoing TKI therapy. However, its positive effects on muscle strength do not seem to be associated with positive effects on fatigue or quality of life. Future studies should therefore compare whether home-based training is more beneficial for patient-reported outcomes. TRIAL REGISTRATION: NCT01645150.

Enterovirus 71 encephalomyelitis and Japanese encephalitis can be distinguished by topographic distribution of inflammation and specific intraneuronal detection of viral antigen and RNA.

AIMS: To investigate if two important epidemic viral encephalitis in children, Enterovirus 71 (EV71) encephalomyelitis and Japanese encephalitis (JE) whose clinical and pathological features may be nonspecific and overlapping, could be distinguished. METHODS: Tissue sections from the central nervous system of infected cases were examined by light microscopy, immunohistochemistry and in situ hybridization. RESULTS: All 13 cases of EV71 encephalomyelitis collected from Asia and France invariably showed stereotyped distribution of inflammation in the spinal cord, brainstem, hypothalamus, cerebellar dentate nucleus and, to a lesser extent, cerebral cortex and meninges. Anterior pons, corpus striatum, thalamus, temporal lobe, hippocampus and cerebellar cortex were always uninflamed. In contrast, the eight JE cases studied showed inflammation involving most neuronal areas of the central nervous system, including the areas that were uninflamed in EV71 encephalomyelitis. Lesions in both infections were nonspecific, consisting of perivascular and parenchymal infiltration by inflammatory cells, oedematous/necrolytic areas, microglial nodules and neuronophagia. Viral inclusions were absent. CONCLUSIONS: Immunohistochemistry and in situ hybridization assays were useful to identify the causative virus, localizing viral antigens and RNA, respectively, almost exclusively to neurones. The stereotyped distribution of inflammatory lesions in EV71 encephalomyelitis appears to be very useful to help distinguish it from JE.

Novel etodolac analog SDX-308 (CEP-18082) induces cytotoxicity in multiple myeloma cells associated with inhibition of beta-catenin/TCF pathway.

We have reported previously that R-enantiomer of etodolac (R-etodolac), which is under investigation in phase 2 clinical trials in chronic lymphocytic leukemia, induces potent cytotoxicity at clinically relevant concentrations in multiple myeloma (MM) cells. In this study, we demonstrated that SDX-308 (CEP-18082), a novel analog of etodolac, has more potent cytotoxicity than R-etodolac against both MM cell lines and patient MM cells, including tumor cells resistant to conventional (dexamethasone, doxorubicine, melphalan) and novel (bortezomib) therapies. SDX-308-induced cytotoxicity is triggered by caspase-8/9/3 activation and poly (ADP-ribose) polymerase cleavage, followed by apoptosis. SDX-308 significantly inhibits beta-catenin/T-cell factor pathway by inhibiting nuclear translocation of beta-catenin, thereby downregulating transcription and expression of downstream target proteins including myc and survivin. Neither interleukin-6 nor insulin-like growth factor-1 protect against growth inhibition triggered by SDX-308. Importantly, growth of MM cells adherent to bone marrow (BM) stromal cells is also significantly inhibited by SDX-308. Our data therefore indicate that the novel etodolac analog SDX-308 can target MM cells in the BM milieu.

Telomere length identifies two different prognostic subgroups among VH-unmutated B-cell chronic lymphocytic leukemia patients.

Some evidences suggest that telomere restriction fragment length (TRF-L) is an effective indicator of histopathogenesis in B-cell tumors. As histopathogenesis is relevant for B-cell chronic lymphocytic leukemia (B-CLL) prognosis, TRF-L was assessed by Southern blot in 201 patients and compared to variable immunoglobulin heave chain gene mutational status (VH-MS) and to other known prognostic features. Overall survival (OS), time to first treatment (TTFT) and progression-free survival (PFS) were evaluated. Our results indicate the following: (1) TRF-L is heterogeneous among B-CLL patients (median 6014 bp, range 1465-16 762); (2) TRF-L correlates to VH-MS (r(2)=0.1994, P<0.0001) with VH-mutated patients showing long and VH-unmutated short telomeres; however, 41% of VH-unmutated and 5% of VH-mutated patients did not show this correlation and were thus defined as 'discordant'; (3) TRF-L effectively predicts outcome in terms of TTFT, PFS and OS; (4) VH-unmutated discordant patients have a better clinical outcome than VH-unmutated concordant patients (OS P<0.01, PFS P<0.05) and similar to that of VH-mutated patients (OS, PFS P=NS). Compared to VH-unmutated concordant patients, VH-unmutated discordant patients showed no peculiarity in their immunoglobulin rearrangement nor in their flow cytometry or fluorescence in situ hybridization profile. In conclusion, TRF-L can be helpful to refine prognostication of B-CLL patients, particularly those with a VH-unmutated immunoglobulin sequence.

Prolonged survival and low incidence of late toxic sequelae in advanced follicular lymphoma treated with a TBI-free autografting program: updated results of the multicenter consecutive GITMO trial.

This study provides an updated report of the consecutive multicenter Gruppo Italiano Trapianto Midollo Osseo trial employing an intensified, purging-free, total body irradiation-free, high-dose sequential chemotherapy schedule with peripheral blood stem cell autograft (i-HDS) in advanced-stage follicular lymphoma (FL). Special interest has been devoted to late toxicities and outcome in terms of molecular status. Ninety-two untreated FL patients aged

Genetic diversity of the hepatitis C virus: impact and issues in the antiviral therapy.

The hepatitis C Virus (HCV) presents a high degree of genetic variability which is explained by the combination of a lack of proof reading by the RNA dependant RNA polymerase and a high level of viral replication. The resulting genetic polymorphism defines a classification in clades, genotypes, subtypes, isolates and quasispecies. This diversity is known to reflect the range of responses to Interferon therapy. The genotype is one of the predictive parameters currently used to define the antiviral treatment strategy and the chance of therapeutic success. Studies have also reported the potential impact of the viral genetic polymorphism in the outcome of antiviral therapy in patients infected by the same HCV genotype. Both structural and non structural genomic regions of HCV have been suggested to be involved in the Interferon pathway and the resistance to antiviral therapy. In this review, we first detail the viral basis of HCV diversity. Then, the HCV genetic regions that may be implicated in resistance to therapy are described, with a focus on the structural region encoded by the E2 gene and the non-structural genes NS3, NS5A and NS5B. Both mechanisms of the Interferon resistance and of the new antiviral drugs are described in this review.

The AP-1 transcription factor JunB is essential for multiple myeloma cell proliferation and drug resistance in the bone marrow microenvironment.

Despite therapeutic advances, multiple myeloma (MM) remains an incurable disease, predominantly because of the development of drug resistance. The activator protein-1 (AP-1) transcription factor family has been implicated in a multitude of physiologic processes and tumorigenesis; however, its role in MM is largely unknown. Here we demonstrate specific and rapid induction of the AP-1 family member JunB in MM cells when co-cultured with bone marrow stromal cells. Supporting a functional key role of JunB in MM pathogenesis, knockdown of JUNB significantly inhibited in vitro MM cell proliferation and survival. Consistently, induced silencing of JUNB markedly decreased tumor growth in a murine MM model of the microenvironment. Subsequent gene expression profiling revealed a role for genes associated with apoptosis, DNA replication and metabolism in driving the JunB-mediated phenotype in MM cells. Importantly, knockdown of JUNB restored the response to dexamethasone in dexamethasone-resistant MM cells. Moreover, 4-hydroxytamoxifen-induced activation of a JunB-ER fusion protein protected dexamethasone-sensitive MM cells against dexamethasone- and bortezomib-induced cytotoxicity. In summary, our results demonstrate for the first time a specific role for AP-1/JunB in MM cell proliferation, survival and drug resistance, thereby strongly supporting that this transcription factor is a promising new therapeutic target in MM.

Clinical significance of cyclooxygenase-2 (COX-2) in multiple myeloma.

Several biological and clinical considerations suggest the involvement of cyclooxygenase-2 (COX-2), the key enzyme of prostaglandin (PG) synthesis, in the pathogenesis and progression of haematological malignancies. Despite the wealth of data concerning COX-2 expression, only limited information is available on multiple myeloma (MM). Using standard immunohistochemistry we therefore evaluated COX-2 protein expression in samples from 57 patients with a primary diagnosis of MM. Time to progression and a variety of clinicopathological features were evaluated by the Kaplan-Meier method and the Cox regression model. In addition, COX-2 expression was evaluated by staining bone marrow from healthy donors and 11 patients with MGUS. Overall, 31 MM samples (54%) expressed COX-2. Positivity for COX-2 was unrelated to stage or clinical or molecular features of the disease. However, patients with COX-2 positive tumours experienced a significantly shorter time to progression (17 vs 30 months, p = 0.037). In summary, COX-2 is frequently expressed in MM and correlates with shorter progression-free survival.

[Pilot epidemiologic study of transmission of cytomegalovirus from mother to preterm infant by breastfeeding]. / Transmission du cytomégalovirus par le lait maternel cru aux enfants prématurés: étude épidémiologique pilote.

UNLABELLED: Transmission of cytomegalovirus (CMV) infection from mothers to preterm infants during breastfeeding may be symptomatic and long term consequences are unknown. This study evaluated the kinetics of CMV load in breastmilk and the rate of postnatal CMV transmission via breastmilk from mothers to their preterm infants. METHODS: Prospective study of mother-child pairs after preterm delivery before 33 weeks. Exclusion of donor breast milk and of CMV-seropositive blood products. Material used was maternal CMV serostatus, ear swab of the infant at birth, weekly screened breast milk and children's urine by rapid viral culture. RESULTS: During a 5-month period 28 mother-infant pairs with 34 preterm infants were studied. Eighteen women (64.3%) were CMV-seronegative at birth; breastmilk samples and the infants' urine remained CMV-negative. Eight of the 10 seropositive mothers, who had 11 preterm infants, excreted CMV into breast milk (80%). CMV excretion into breast milk was detected during the first week after delivery in 66% cases and was at its peaked between 3 to 5 weeks after delivery. Out of the 7 CMV-exposed infants, CMV transmission was confirmed in only one asymptomatic case. Total quantity of breast milk intake did not seem discriminative for CMV transmission. CONCLUSION: In CMV-seropositive mothers of preterm infants a high incidence of CMV excretion into breast milk was detected. Despite this high rate, symptomatic infection did not occur. However, potential risk and severity of infection may be difficult to establish. Because breastfeeding is beneficial, new procedures for gentle virus inactivation of seropositive breast milk should be assessed.

Concurrent administration of high-dose chemotherapy and rituximab is a feasible and effective chemo/immunotherapy for patients with high-risk non-Hodgkin's lymphoma.

The aim of this study was to investigate feasibility, tolerability and efficacy of rituximab-supplemented high-dose sequential chemotherapy (R-HDS) with peripheral blood progenitor cell autografting as frontline or salvage treatment in patients with advanced non-Hodgkin's lymphoma (NHL). Thirty-two patients have been treated: 14 at disease onset and 18 with relapsed or progressive disease. R-HDS regimens included six courses of rituximab. Rituximab was delivered either concurrently with high-dose chemotherapy to exploit the in vivo purging properties of the drug as well as at the end of the treatment plan to target minimal residual disease. All patients treated at disease onset completed their treatment with no life-threatening toxicity, while two toxic deaths due to severe bilateral pneumonia were observed among patients treated due to relapsed or refractory disease. Thirteen of 14 patients treated up-front achieved CR. Among pre-treated patients 10 of 18 achieved CR with better results in patients with relapsed (seven of eight) compared to progressive disease (three of 10). PCR analysis was carried out in indolent lymphoma patients: nine of nine follicular lymphomas and three of six CD5-positive NHL collected PCR-negative peripheral blood progenitor cell harvests. The results of this pilot study show that R-HDS is feasible and effective with acceptable toxicity when used at disease onset. In pre-treated patients this treatment also showed promising results, although the risk of severe infections needs to be considered.

Identification of a duplicated V3 domain in NS5A associated with cirrhosis and hepatocellular carcinoma in HCV-1b patients.

BACKGROUND: The NS5A protein of the hepatitis C virus has been shown to be involved in the development of hepatocellular carcinoma. OBJECTIVES: In a French multicenter study, we investigated the clinical and epidemiological features of a new HCV genotype 1b strain bearing a wide insertion into the V3 domain. STUDY DESIGN: We studied NS5A gene sequences in 821 French patients infected with genotype 1b HCV. RESULTS: We identified an uncharacterized V3 insertion without ORF disruption in 3.05% of the HCV sequences. The insertion comprised 31 amino-acids for the majority of patients; 3 patients had 27 amino-acids insertions and 1 had a 12 amino-acids insertion. Sequence identity between the 31 amino-acids insertions and the V3 domain ranged from 48 to 96% with E-values above 4e(-5), thus illustrating sequence homology and a partial gene duplication event that to our knowledge has never been reported in HCV. Moreover we showed the presence of the duplication at the time of infection and its persistence at least during 12 years in the entire quasispecies. No association was found with extrahepatic diseases. Conversely, patients with cirrhosis were two times more likely to have HCV with this genetic characteristic (p=0.04). Moreover, its prevalence increased with liver disease severity (from 3.0% in patients without cirrhosis to 9.4% in patients with both cirrhosis and HCC, p for trend=0.045). CONCLUSIONS: We identified a duplicated V3 domain in the HCV-1b NS5A protein for the first time. The duplication may be associated with unfavorable evolution of liver disease including a possible involvement in liver carcinogenesis.

Coronavirus-related nosocomial viral respiratory infections in a neonatal and paediatric intensive care unit: a prospective study.

The incidence of nosocomial viral respiratory infections (NVRI) in neonates and children hospitalized in paediatric and neonatal intensive care units (PNICU) is unknown. Human coronaviruses (HCoV) have been implicated in NVRI in hospitalized preterm neonates. The objectives of this study were to determine the incidence of HCoV-related NVRI in neonates and children hospitalized in a PNICU and the prevalence of viral respiratory tract infections in staff. All neonates (age< or =28 days) and children (age>28 days) hospitalized between November 1997 and April 1998 were included. Nasal samples were obtained by cytological brush at admission and weekly thereafter. Nasal samples were taken monthly from staff. Virological studies were performed, using indirect immunofluorescence, for HCoV strains 229E and OC43, respiratory syncytial virus (RSV), influenza virus types A and B, paramyxoviruses types 1, 2 and 3 and adenovirus. A total of 120 patients were enrolled (64 neonates and 56 children). Twenty-two samples from 20 patients were positive (incidence 16.7%). In neonates, seven positive samples, all for HCoV, were detected (incidence 11%). Risk factors for NVRI in neonates were: duration of hospitalization, antibiotic treatment and duration of parenteral nutrition (P<0.01). Monthly prevalence of viral infections in staff was between 0% and 10.5%, mainly with HCoV. In children, 15 samples were positive in 13 children at admission (seven RSV, five influenza and three adenovirus) but no NVRI were observed. In spite of a high rate of community-acquired infection in hospitalized children, the incidence of NVRI with common respiratory viruses appears low in neonates, HCoV being the most important pathogen of NRVI in neonates during this study period. Further research is needed to evaluate the long-term impact on pulmonary function.

Probiotics: a new way to fight bacterial pulmonary infections?

Antibiotics, of which Fleming has identified the first representative, penicillin, in 1928, allowed dramatical improvement of the treatment of patients presenting with infectious diseases. However, once an antibiotic is used, resistance may develop more or less rapidly in some bacteria. It is thus necessary to develop therapeutic alternatives, such as the use of probiotics, defined by the World Health Organization (WHO) as "micro-organisms which, administered live and in adequate amounts, confer a benefit to the health of the host". The scope of these micro-organisms is broad, concerning many areas including that of infectious diseases, especially respiratory infections. We describe the rational use of probiotics in respiratory tract infections and detail the results of various clinical studies describing the use of probiotics in the management of respiratory infections such as nosocomial or community acquired pneumonia, or on specific grounds such as cystic fibrosis. The results are sometimes contradictory, but the therapeutic potential of probiotics seems promising. Implementing research to understand their mechanisms of action is critical to conduct therapeutic tests based on a specific rational for the strains to be used, the dose, as well as the chosen mode and rhythm of administration.

Lenalidomide in combination with an activin A-neutralizing antibody: preclinical rationale for a novel anti-myeloma strategy.

Given the prevalence of osteolytic bone disease in multiple myeloma (MM), novel therapies targeting bone microenvironment are essential. Previous studies have identified activin A to be of critical importance in MM-induced osteolysis. Lenalidomide is a known and approved treatment strategy for relapsed MM. Our findings demonstrate that lenalidomide acts directly on bone marrow stromal cells via an Akt-mediated increase in Jun N-terminal kinase-dependent signaling resulting in activin A secretion, with consequent inhibition of osteoblastogenesis. Here, we attempted to augment the antitumor benefits of lenalidomide while overcoming its effects on osteoblastogenesis by combining it with a neutralizing antibody to activin A. Increased activin A secretion induced by lenalidomide was abrogated by the addition of activin A-neutralizing antibody, which effectively restored osteoblast function and inhibited MM-induced osteolysis without negating the cytotoxic effects of lenalidomide on malignant cells. This provides the rationale for an ongoing clinical trial (NCT01562405) combining lenalidomide with an anti-activin A strategy.

A novel role for CCL3 (MIP-1α) in myeloma-induced bone disease via osteocalcin downregulation and inhibition of osteoblast function.

Upregulation of cytokines and chemokines is a frequent finding in multiple myeloma (MM). CCL3 (also known as MIP-1α) is a pro-inflammatory chemokine, levels of which in the MM microenvironment correlate with osteolytic lesions and tumor burden. CCL3 and its receptors, CCR1 and CCR5, contribute to the development of bone disease in MM by supporting tumor growth and regulating osteoclast (OC) differentiation. In this study, we identify inhibition of osteoblast (OB) function as an additional pathogenic mechanism in CCL3-induced bone disease. MM-derived and exogenous CCL3 represses mineralization and osteocalcin production by primary human bone marrow stromal cells and HS27A cells. Our results suggest that CCL3 effects on OBs are mediated by ERK activation and subsequent downregulation of the osteogenic transcription factor osterix. CCR1 inhibition reduced ERK phosphorylation and restored both osterix and osteocalcin expression in the presence of CCL3. Finally, treating SCID-hu mice with a small molecule CCR1 inhibitor suggests an upregulation of osteocalcin expression along with OC downregulation. Our results show that CCL3, in addition to its known catabolic activity, reduces bone formation by inhibiting OB function, and therefore contributes to OB/OC uncoupling in MM.

AT7519, A novel small molecule multi-cyclin-dependent kinase inhibitor, induces apoptosis in multiple myeloma via GSK-3beta activation and RNA polymerase II inhibition.

Dysregulated cell cycling is a universal hallmark of cancer and is often mediated by abnormal activation of cyclin-dependent kinases (CDKs) and their cyclin partners. Overexpression of individual complexes are reported in multiple myeloma (MM), making them attractive therapeutic targets. In this study, we investigate the preclinical activity of a novel small-molecule multi-CDK inhibitor, AT7519, in MM. We show the anti-MM activity of AT7519 displaying potent cytotoxicity and apoptosis; associated with in vivo tumor growth inhibition and prolonged survival. At the molecular level, AT7519 inhibited RNA polymerase II (RNA pol II) phosphorylation, a CDK9, 7 substrate, associated with decreased RNA synthesis confirmed by [(3)H] Uridine incorporation. In addition, AT7519 inhibited glycogen synthase kinase 3beta (GSK-3beta) phosphorylation; conversely pretreatment with a selective GSK-3 inhibitor and shRNA GSK-3beta knockdown restored MM survival, suggesting the involvement of GSK-3beta in AT7519-induced apoptosis. GSK-3beta activation was independent of RNA pol II dephosphorylation confirmed by alpha-amanitin, a specific RNA pol II inihibitor, showing potent inhibition of RNA pol II phosphorylation without corresponding effects on GSK-3beta phosphorylation. These results offer new insights into the crucial, yet controversial role of GSK-3beta in MM and show significant anti-MM activity of AT7519, providing the rationale for its clinical evaluation in MM.