Clofazimine for Treatment of Cryptosporidiosis in Human Immunodeficiency Virus Infected Adults: An Experimental Medicine, Randomized, Double-blind, Placebo-controlled Phase 2a Trial.

BACKGROUND: We evaluated the efficacy, pharmacokinetics (PK), and safety of clofazimine (CFZ) in patients living with human immunodeficiency virus (HIV) with cryptosporidiosis. METHODS: We performed a randomized, double-blind, placebo-controlled study. Primary outcomes in part A were reduction in Cryptosporidium shedding, safety, and PK. Primary analysis was according to protocol (ATP). Part B of the study compared CFZ PK in matched individuals living with HIV without cryptosporidiosis. RESULTS: Twenty part A and 10 part B participants completed the study ATP. Almost all part A participants had high viral loads and low CD4 counts, consistent with failure of antiretroviral (ARV) therapy. At study entry, the part A CFZ group had higher Cryptosporidium shedding, total stool weight, and more diarrheal episodes compared with the placebo group. Over the inpatient period, compared with those who received placebo, the CFZ group Cryptosporidium shedding increased by 2.17 log2 Cryptosporidium per gram stool (95% upper confidence limit, 3.82), total stool weight decreased by 45.3 g (P = .37), and number of diarrheal episodes increased by 2.32 (P = .87). The most frequent solicited adverse effects were diarrhea, abdominal pain, and malaise. One placebo and 3 CFZ participants died during the study. Plasma levels of CFZ in participants with cryptosporidiosis were 2-fold lower than in part B controls. CONCLUSIONS: Our findings do not support the efficacy of CFZ for the treatment of cryptosporidiosis in a severely immunocompromised HIV population. However, this trial demonstrates a pathway to assess the therapeutic potential of drugs for cryptosporidiosis treatment. Screening persons living with HIV for diarrhea, and especially Cryptosporidium infection, may identify those failing ARV therapy. CLINICAL TRIALS REGISTRATION: NCT03341767.

Increased intracellular survival of Salmonella Typhimurium ST313 in HIV-1-infected primary human macrophages is not associated with Salmonella hijacking the HIV compartment.

BACKGROUND: Non-typhoidal Salmonella (NTS) causes a severe invasive syndrome (iNTS disease) described in HIV-positive adults. The impact of HIV-1 on Salmonella pathogenesis and the molecular basis for the differences between these bacteria and classical diarrhoeal S. Typhimurium remains unclear. RESULTS: Here, we show that iNTS-associated S. Typhimurium Sequence Type 313 (ST313) bacteria show greater intracellular survival in primary human macrophages, compared with a 'classical' diarrhoeal S. Typhimurium ST19 isolate. The increased intracellular survival phenotype of ST313 is more pronounced in HIV-infected macrophages. We explored the possibility that the bacteria take advantage of the HIV-associated viral-containing compartments created in human macrophages that have low pH. Confocal fluorescence microscopy and focussed ion beam-scanning electron microscopy tomography showed that Salmonella did not co-localise extensively with HIV-positive compartments. CONCLUSION: The capacity of ST313 bacteria to survive better than ST19 bacteria within primary human macrophages is enhanced in cells pre-infected with HIV-1. Our results indicate that the ST313 bacteria do not directly benefit from the niche created by the virus in HIV-1-infected macrophages, and that they might take advantage from a more globally modified host cell. SIGNIFICANCE: A better understanding of the interplay between HIV-1 and Salmonella is important not only for these bacteria but also for other opportunistic pathogens.

Soil organic matter and the extracellular microbial matrix show contrasting responses to C and N availability.

An emerging paradigm in soil science suggests microbes can perform 'N mining' from recalcitrant soil organic matter (SOM) in conditions of low N availability. However, this requires the production of extracellular structures rich in N (including enzymes and structural components) and thus defies stoichiometric expectation. We set out to extract newly synthesised peptides from the extracellular matrix in soil and compare the amino acid (AA) profiles, N incorporation and AA dynamics in response to labile inputs of contrasting C/N ratio. Glycerol was added both with and without an inorganic source of N (10% 15N labelled NH4NO3) to a soil already containing a large pool of refractory SOM and incubated for 10 days. The resulting total soil peptide (TSP) and extracellular pools were compared using colorimetric methods, gas chromatography, and isotope ratio mass spectrometry. N isotope compositions showed that the extracellular polymeric substance (EPS) contained a greater proportion of products formed de novo than did TSP, with hydrophobic EPS-AAs (leucine, isoleucine, phenylalanine, hydroxyproline and tyrosine) deriving substantially more N from the inorganic source provided. Quantitative comparison between extracts showed that the EPS contained greater relative proportions of alanine, glycine, proline, phenylalanine and tyrosine. The greatest increases in EPS-peptide and EPS-polysaccharide concentrations occurred at the highest C/N ratios. All EPS-AAs responded similarly to treatment whereas the responses of TSP were more complex. The results suggest that extracellular investment of N (as EPS peptides) is a microbial survival mechanism in conditions of low N/high C which, from an evolutionary perspective, must ultimately lead to the tendency for increased N returns to the microbial biomass. A conceptual model is proposed that describes the dynamics of the extracellular matrix in response to the C/N ratio of labile inputs.

Assessment of HER2 gene amplification in adenocarcinomas of the stomach or gastroesophageal junction in the INT-0116/SWOG9008 clinical trial.

BACKGROUND: Trastuzumab has been approved for patients with human epidermal growth factor receptor 2 (HER2)-positive metastatic gastric carcinoma; however, relatively little is known about the role of HER2 in the natural history of this disease. PATIENTS AND METHODS: Patients enrolled in the INT-0116/SWOG9008 phase III gastric cancer clinical trial with available tissue specimens were retrospectively evaluated for HER2 gene amplification by FISH and overexpression by immunohistochemistry (IHC). The original trial was designed to evaluate the benefit of postoperative chemoradiation compared with surgery alone. RESULTS: HER2 gene amplification rate by FISH was 10.9% among 258 patients evaluated. HER2 overexpression rate by IHC was 12.2% among 148 patients evaluated, with 90% agreement between FISH and IHC. There was a significant interaction between HER2 amplification and treatment with respect to both disease-free survival (DFS) (P = 0.020) and overall survival (OS) (P = 0.034). Among patients with HER2-non-amplified cancers, treated patients had a median OS of 44 months compared with 24 months in the surgery-only arm (P = 0.003). Among patients with HER2-amplified cancers, there was no significant difference in survival based on treatment arm. HER2 status was not a prognostic marker among patients who received no postoperative chemoradiation. CONCLUSION: Patients lacking HER2 amplification benefited from treatment as indicated by both DFS and OS. CLINICAL TRIAL: INT-0116/SWOG9008 phase III.

A comparison of two colorimetric assays, based upon Lowry and Bradford techniques, to estimate total protein in soil extracts.

Soil extracts usually contain large quantities of dissolved humified organic material, typically reflected by high polyphenolic content. Since polyphenols seriously confound quantification of extracted protein, minimising this interference is important to ensure measurements are representative. Although the Bradford colorimetric assay is used routinely in soil science for rapid quantification protein in soil-extracts, it has several limitations. We therefore investigated an alternative colorimetric technique based on the Lowry assay (frequently used to measure protein and humic substances as distinct pools in microbial biofilms). The accuracies of both the Bradford assay and a modified Lowry microplate method were compared in factorial combination. Protein was quantified in soil-extracts (extracted with citrate), including standard additions of model protein (BSA) and polyphenol (Sigma H1675-2). Using the Lowry microplate assay described, no interfering effects of citrate were detected even with concentrations up to 5 times greater than are typically used to extract soil protein. Moreover, the Bradford assay was found to be highly susceptible to two simultaneous and confounding artefacts: 1) the colour development due to added protein was greatly inhibited by polyphenol concentration, and 2) substantial colour development was caused directly by the polyphenol addition. In contrast, the Lowry method enabled distinction between colour development from protein and non-protein origin, providing a more accurate quantitative analysis. These results suggest that the modified-Lowry method is a more suitable measure of extract protein (defined by standard equivalents) because it is less confounded by the high polyphenolic content which is so typical of soil extracts.

A let-7 microRNA-binding site polymorphism in 3'-untranslated region of KRAS gene predicts response in wild-type KRAS patients with metastatic colorectal cancer treated with cetuximab monotherapy.

PURPOSE: recent studies have found that KRAS mutations predict resistance to monoclonal antibodies targeting the epidermal growth factor receptor in metastatic colorectal cancer (mCRC). A polymorphism in a let-7 microRNA complementary site (lcs6) in the KRAS 3' untranslated region (UTR) is associated with an increased cancer risk in non-small-cell lung cancer and reduced overall survival (OS) in oral cancers. We tested the hypothesis whether this polymorphism may be associated with clinical outcome in KRAS wild-type (KRASwt) mCRC patients treated with cetuximab monotherapy. PATIENTS AND METHODS: the presence of KRAS let-7 lcs6 polymorphism was evaluated in 130 mCRC patients who were enrolled in a phase II study of cetuximab monotherapy (IMCL-0144). Genomic DNA was extracted from dissected formalin-fixed paraffin-embedded tumor tissue, KRAS mutation status and polymorphism were assessed using direct sequencing and PCR restriction fragment length polymorphism technique. RESULTS: KRAS let-7 lcs6 polymorphism was found to be related to object response rate (ORR) in mCRC patients whose tumors had KRASwt. The 12 KRASwt patients harboring at least a variant G allele (TG or GG) had a 42% ORR compared with a 9% ORR in 55 KRASwt patients with let-7 lcs6 TT genotype (P = 0.02, Fisher's exact test). KRASwt patients with TG/GG genotypes had trend of longer median progression-free survival (3.9 versus 1.3 months) and OS (10.7 versus 6.4 months) compared to those with TT genotypes. CONCLUSIONS: these results are the first to indicate that the KRAS 3'UTR polymorphism may predict for cetuximab responsiveness in KRASwt mCRC patients, which warrants validation in other clinical trials.

Gender-related survival differences associated with polymorphic variants of estrogen receptor-ß (ERß) in patients with metastatic colon cancer.

Estrogen replacement therapy in women has shown a protective effect on the development of colonic carcinomas. Gender-related differences in the development of colonic carcinomas have also been reported. Estrogen receptor-ß (ERß) is expressed in colon carcinomas and has shown prognostic value in colon cancer patients. This study investigated an ERß 3' non-coding polymorphism associated with transcriptional activity to determine clinical outcome in patients with metastatic colon cancer. Genomic DNA from 318 metastatic colon cancer patients, 177 males and 141 females, were collected from 1992 to 2003. These patients were analyzed for CA repeat polymorphism of the ERß gene. Gender-related survival differences were associated with an ERß (CA)n repeat polymorphism (P for interaction=0.003, the likelihood ratio test). Female patients with any short<22 (CA)n repeat alleles had shorter overall survival (OS) compared with female patients who had both long≥22 (CA)n repeat alleles. In the male patients, the opposite OS difference was found. This study supports the role of an ERß (CA)n repeat polymorphism as a prognostic marker in metastatic colon cancer; however, this prognostic factor had opposite implications based on gender.

Interstellar sulfur hydride: a search for the 111-megahertz lines.

Similarities in the energy-level structure of the sulfur hydride radical and the hydroxyl radical suggest that sulfur hydride in the interstellar medium might be detectable because of a population inversion or anti-inversion similar to that of the hydroxyl radical. We have searched for the 111.54-megahertz transition [F (total angular momentum quantum number) = 2 --> 2] and for the 111.22-megahertz transition (F = 1 --> 1) in the galactic radio source W49, one of the brightest hydroxyl emission sources. No sulfur hydride emission lines with half-power widths of 130 hertz or greater were detected with the 1000-foot Arecibo antenna. The upper limits established with 100-hertz filters were 50 and 60 flux units (1 flux unit= 10(26) watt meter(-2) hertz(-1)), respectively, for the two lines.

Nonspecific effects of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a.

Epidemiological and immunological evidence suggests that some vaccines can reduce all-cause mortality through nonspecific changes made to innate immune cells. Here, we present the first data to describe the nonspecific immunological impact of oral vaccination with live-attenuated Salmonella Typhi strain Ty21a. We vaccinated healthy adults with Ty21a and assessed aspects of innate and adaptive immunity over the course of 6 months. Changes to monocyte phenotype/function were observed for at least 3 months. Changes to innate and adaptive immune cell cytokine production in response to stimulation with vaccine and unrelated nonvaccine antigens were observed over the 6-month study period. The changes that we have observed could influence susceptibility to infection through altered immune responses mounted to subsequently encountered pathogens. These changes could influence all-cause mortality.

Polymorphisms in VEGF and IL-8 predict tumor recurrence in stage III colon cancer.

BACKGROUND: Identifying molecular markers for tumor recurrence is critical in successfully selecting patients with stage III colon cancer who are more likely to benefit from adjuvant chemotherapy. The present study analyzed a subset of 10 polymorphisms within eight genes involved in the tumor angiogenesis pathway and their impact on prognosis in stage III colon cancer patients treated with adjuvant chemotherapy. PATIENTS AND METHODS: Blood samples were obtained from 125 patients with locally advanced colon cancer at University of Southern California medical facilities. DNA was extracted from peripheral blood and the genotypes were analyzed using PCR-restriction fragment length polymorphism and 5'-end [gamma-(33)P] ATP-labeled PCR protocols. RESULTS: Polymorphisms in vascular endothelial growth factor (VEGF) (C+936T; P = 0.003, log-rank test) and interleukin-8 (IL-8) (T-251A; P = 0.04, log-rank test) were independently associated with risk of recurrence in stage III colon cancer patients. In combined analysis, grouping alleles into favorable versus nonfavorable alleles, high expression variants of VEGF C+936T and IL-8 T-251A were associated with a higher likelihood of developing tumor recurrence (P < 0.001). CONCLUSION: High expression variants of VEGF C+936T and IL-8 T-251A were associated with shorter time to tumor recurrence, indicating that the analysis of angiogenesis-related gene polymorphisms may help to identify patient subgroups at high risk for tumor recurrence.