Clinical and molecular insights into BCG immunotherapy for melanoma.

The incidence of cutaneous melanoma and the mortality rate of advanced melanoma patients continue to rise globally. Despite the recent success of immunotherapy including ipilimumab and pembrolizumab checkpoint inhibitors, a large proportion of patients are refractory to such treatment modalities. The application of mycobacteria such as Bacillus Calmette-Guérin (BCG) in the treatment of various malignancies, including cutaneous melanoma, has been clearly demonstrated after almost a century of observations and experimentation. Intralesional BCG (IL-BCG) immunotherapy is a highly efficient and cost-effective treatment option for inoperable stage III in-transit melanoma, as recommended in the National Comprehensive Cancer Network Guidelines. IL-BCG has shown great efficacy in the regression of directly injected metastatic melanoma lesions, as well as distal noninjected nodules in immunocompetent patients. Clinical and preclinical studies have shown that BCG serves as a strong immune modulator, inducing the recruitment of various immune cells that contribute to antitumour immunity. However, the specific mechanism of BCG-mediated tumour immunity remains poorly understood. Comparative genome analyses have revealed that different BCG strains exhibit distinct immunological activity and virulence, which might impact the therapeutic response and clinical outcome of patients. In this review, we discuss the immunostimulatory potential of different BCG substrains and highlight clinical studies utilizing BCG immunotherapy for the treatment of cutaneous melanoma. Furthermore, the review focuses on the cellular and molecular mechanisms of the BCG-induced immune responses of both the innate and adaptive arms of the immune system. Furthermore, the review discussed the administration of BCG as a monotherapy or in combination with other immunotherapeutic or chemotherapeutic agents.

The combined effect of bacteria and Chlorella vulgaris on the treatment of municipal wastewaters.

Impacts of Chlorella vulgaris with or without co-existing bacteria on the removal of nitrogen, phosphorus and organic matter from wastewaters were studied by comparing the wastewater treatment effects between an algae-bacteria consortium and a stand-alone algae system. In the algae-bacteria system, C.vulgaris played a dominant role in the removal of nitrogen and phosphorus, while bacteria removed most of the organic matter from the wastewater. When treating unsterilized wastewater, bacteria were found to inhibit the growth of algae at >231 mg/L dissolved organic carbon (DOC). Using the algae-bacteria consortium resulted in the removal of 97% NH4(+), 98% phosphorus and 26% DOC at a total nitrogen (TN) level of 29-174 mg/L. The reaction rate constant (k) values in sterilized and unsterilized wastewaters were 2.17 and 1.92 mg NH4(+)-N/(mg algal cell ·d), respectively.

Injection of an alpha-melanocyte stimulating hormone expression plasmid is effective in suppressing experimental autoimmune uveitis.

PURPOSE: The neuropeptide, alpha-melanocyte stimulating hormone (alpha-MSH), is an endogenous antagonist of inflammation. Injections of alpha-MSH peptide into inflamed tissues have been found to be very effective in suppressing autoimmune and endotoxin mediated diseases. We evaluated the potential to suppress ocular autoimmune disease (uveitis) by augmenting the expression of alpha-MSH through subconjunctival injections of naked adrenocorticotropic hormone amino acids 1-17 (ACTH1-17) plasmid. METHODS: We clinically scored the uveitis over time in B10.RIII, C57BL/6, and melanocortin 5 receptor knock-out (MC5r((-/-))) mice with experimental autoimmune uveitis (EAU) that were conjunctively injected with a naked DNA plasmid encoding ACTH1-17 at the time of EAU onset and three days later. The post-EAU retina histology of plasmid injected eyes was examined, and post-EAU concentrations of alpha-MSH in aqueous humor was assayed by ELISA. RESULTS: The subconjunctival injection of ACTH1-17 plasmid augmented the concentration of alpha-MSH in the aqueous humor of all post-EAU mice. The injection of ACTH1-17 suppressed the severity of EAU in the B10.RIII and C57BL/6 mice but the MC5r((-/-)) mice. In all the models of EAU, the ACTH1-17 injection helped to preserve the structural integrity of the retina; however, post-EAU aqueous humor was not immunosuppressive. CONCLUSIONS: The subconjunctival injection of the alpha-MSH expression vector ACTH1-17 plasmid is effective in suppressing EAU. The suppressive activity is dependent on MC5r expression, and possibly works though alpha-MSH antagonism of inflammation than on alpha-MSH directly modulating immune cells. The results suggest that an effective therapy for uveitis could include a gene therapy approach based on delivering alpha-MSH.

Squamous cell granulomas of the neck: histologic regression of metastatic squamous cell carcinoma following chemotherapy and/or radiotherapy.

BACKGROUND: For patients with squamous cell carcinoma of the head and neck (HNSCC), persistence of cervical adenopathy following organ-preservation therapy is a strong predictor of locoregional failure. Squamous cell granulomas of the neck may represent a regressed state of metastatic HNSCC; however, relevant clinicopathologic features of this lesion including its morphologic characteristics, association with therapy, and relationship to disease progression are not well defined. METHODS: We reviewed 866 consecutive neck dissections performed at The Johns Hopkins Hospital from 1984 to 1996. A total of eight cases showing a foreign-body giant-cell reaction to keratin in the absence of viable tumor formed the basis of this study. RESULTS: All eight cases were from patients with stage III or IV HNSCC with concurrent neck masses. Patients were initially treated by chemotherapy (n = 1), radiotherapy (n = 1), or chemotherapy plus radiotherapy (n = 6); and all patients subsequently underwent neck dissection for persistence of their neck masses. Histologically, the neck lesions were characterized by a foreign-body giant-cell reaction to keratin and extensive scarring. None (0%) of the patients developed recurrent regional disease in the treated neck. Two (25%) of the patients had tumor recurrence at the primary site. Two (25%) of the patients developed widely metastatic disease. CONCLUSIONS: These observations suggest that squamous cell granulomas represent histologic regression of metastatic squamous cell carcinoma in patients with HNSCC treated by chemotherapy and/or radiotherapy. Although persistent cervical adenopathy is an established risk factor for locoregional failure in this group of patients, squamous cell granulomas of the neck paradoxically may reflect enhanced regional tumor sensitivity to cytotoxic agents.

CT-simulator based brachytherapy planner: seed localization and incorporation of biological considerations.

Radiation dose prescription, interpretation, and planning can be problematic for brachytherapy due to high spatial heterogeneity, varying and various dose rates, absence of superimposed calculated isodose distributions onto affected tissues, and lack of dose volume histograms. A new treatment planner has been developed to reduce these limitations in brachytherapy planning. The PC-based planning system uses a CT-simulator to sequentially scan the patient to generate orthogonal images (to localize seed positions) and subsequently axially scan the patient. This sequential scanning procedure avoids using multiple independent patient scans, templates, external frames, or fiducial markers to register the reconstructed seed positions with patient contours. Dose is computed after assigning activity to (low dose rate) Ir192, linear Cs137, or I125 seeds or dwell times (high dose rate) to the Ir192 source. The planar isodose distribution is superimposed onto axial, coronal, or sagittal views of the tissues following image reconstruction. The treatment plan computes (1) direct and cumulative volume dose histograms for individual tissues, (2) the average, standard deviation, and coefficient of skewness of the dose distribution within individual tissues, (3) an average (over all tissue pixels) survival probability (S) and average survival dose DASD for a given radiation treatment, (4) normal tissue complication probability (NTCP) delivered to a given tissue. All four computed quantities account for dose heterogeneity. These estimates of the biological response to radiation from laboratory-based studies may help guide the evaluation of the prescribed low- or high-dose rate therapy in retrospective and prospective clinical studies at a number of treatment sites.

Hypoxic sensitizer and cytotoxin for head and neck cancer.

Tumour hypoxia is well recognised as a major factor contributing to radioresistance. This article examines the role of hypoxia in influencing the treatment outcome following radiotherapy (RT), and reviews the rationale and results of clinical trials that utilise hypoxic sensitizers or cytotoxins in the treatment of head and neck carcinoma. Histologic evidence for tumour hypoxia in human neoplasms was first reported in 1955. Since then, direct measurement by microelectrodes has revealed heterogeneity in intratumoural oxygen concentrations, and low oxygen concentrations are associated with poor local-regional control by RT. These findings coupled with the result of nuclear imaging studies employing radiolabelled imidazoles, provide strong evidence for the existence of tumour hypoxia which influences RT treatment outcome. Hyperbaric oxygen (HBO) trials for head and neck cancer, conducted in the early 1970s, demonstrated that HBO improved local control and survival rates in patients with head and neck cancer receiving radiotherapy (RT). Since the mid-1970s, clinical research in overcoming tumour hypoxia was mainly centred on the use of nitro-imidazoles as hypoxic cell sensitizers. However, the results from several major clinical trials remain inconclusive. Specifically, the Radiation Therapy Oncology Group (RTOG) misonidazole head and neck trial (298 patients) showed no benefit. The Danish misonidazole trial (626 patients) showed no overall benefit, however positive results were observed in a subgroup (304 pharyngeal cancer patients). Although the European Organisation for Research and Teaching of Cancer (EORTC) misonidazole trial with hyperfractionated RT showed no benefit, the Danish nimorazole trial demonstrated an overall benefit in survival as well as local control. The European etanidazole (ETA) trial (374 patients) showed no advantage of adding the drug to RT. The RTOG ETA trial (504 patients) showed no global benefit. However, positive results were observed in a subset of patients with early nodal disease (197 patients). In addition, a recent meta-analysis by Overgaard, utilising pooled results in the literature demonstrated that modification of tumour hypoxia significantly improved local-regional control in head and neck cancers with an odds ratio of 1.23 (95% confidence limits 1.09 to 1.37). Hypoxic cytotoxins, such as tirapazamine, represent a novel approach in overcoming radioresistant hypoxic cells. Tirapazamine is a bioreductive agent which, by undergoing one electron reduction in hypoxic conditions, forms cytotoxic free radicals that produce DNA strand breaks causing cell death. In vitro and in vivo laboratory studies demonstrate that tirapazamine is 40 to 150 times more toxic to cells under hypoxic conditions as compared to oxygenated conditions and that tirapazamine is superior to ETA in enhancing fractionated irradiation in mouse SCCVII and other tumour types with an enhancement ratio of 1.5 to 3.0. Phase I studies demonstrated that therapeutic doses of tirapazamine can be given safely. A multi-institutional phase II trial using tirapazamine with concurrent RT for head and neck cancer is now in progress.

Psychosocial correlates of immune responsiveness and illness episodes in US Air Force Academy cadets undergoing basic cadet training.

This study examined psychosocial correlates of immune function and illness in 89 male first-year US Air Force Academy cadets. A psychosocial questionnaire was administered to cadets prior to their arrival at the academy and was readministered during cadet orientation and during the stressful environment of Basic Cadet Training (BCT). Immune responsiveness was analyzed by PHA-, PMA-, or anti-CD3-stimulated thymidine uptake in mononuclear leucocytes. Illness episodes were assessed via medical chart review and self-reported symptoms. There were significant increases in distress levels as cadets entered BCT. No psychosocial measure assessed prior to arrival at the academy predicted level of PHA-, PMA-, and anti-CD3-stimulated thymidine uptake or risk of illness. However, hostility levels reported during BCT predicted risk of illness in the four weeks following psychosocial assessment (odds ratio = 7.1; 95% confidence interval: 1.4-36.1). Elevated response to environmental stressors and lower well-being levels also predicted impending illness, but only in the cohort of cadets who had not contracted food poisoning prior to assessment during BCT (OR = 9.3, CI = 1.9-46.7; OR = 0.09, CI = 0.02-0.53). These results suggest that self-report measures of hostility, response to environmental stressors and well-being may be useful predictors of impending illness episodes in males encountering high stress environments.

Mutations in human cytomegalovirus UL97 gene confer clinical resistance to ganciclovir and can be detected directly in patient plasma.

Specific mutations in the UL97 region of human cytomegalovirus (HCMV) have been found to confer resistance to laboratory-adapted strains subjected to ganciclovir selection. In this study, mutations in the UL97 region of HCMV isolates obtained from patients receiving ganciclovir therapy were examined to determine whether they would confer ganciclovir resistance, and if these mutations could be detected directly in the plasma of AIDS patients with progressive HCMV disease despite ganciclovir treatment. A single nucleotide change within a conserved region of UL97 was found in five resistant isolates, resulting in an amino acid substitution in residue 595: from leucine to phenylalanine in one, and from leucine to serine in four resistant isolates. A sixth resistant isolate demonstrated a single nucleotide change, leading to a threonine to isoleucine substitution in residue 659. The role of the 595 amino acid substitution in conferring ganciclovir resistance was confirmed by marker transfer experiments. In further studies, direct sequencing of HCMV DNA present in plasma obtained from persons with resistant viruses revealed the identical amino acid substitutions in plasma as those present in the cultured viruses. These findings indicate that clinical resistance to ganciclovir can result from specific point mutations in the UL97 gene, and that the emergence of the resistant genotype can be detected directly in patient plasma.

Health behaviors, risk factors, and health indicators associated with cigarette use in Mexican Americans: results from the Hispanic HANES.

BACKGROUND: Cigarette smokers often engage in other, potentially deleterious, health behaviors. Such behaviors have not been well documented in Mexican American smokers. METHODS: Data from the Southwestern sample of the Hispanic Health and Nutrition Examination Survey (HHANES) were employed to investigate differences in health behaviors, risk factors and health indicators between cigarette smokers and nonsmokers among Mexican Americans. Differences between those smoking less than 10 and 10 or more cigarettes per day were also examined by age group and gender. RESULTS: Positive associations between smoking status and heavy coffee and alcohol consumption were found across gender and age groups. Less consistent was the finding that smokers weighed less than nonsmokers. Lower systolic and diastolic blood pressures in middle-aged smokers, and higher levels of depressive symptomatology among smoking women were found. Those smoking 10 or more cigarettes per day were more likely to report heavy coffee consumption, with younger men reporting greater activity limitation due to poor health. Middle-aged men and women in the 10+ category were generally in better health than lighter smokers. CONCLUSIONS: Modest associations between cigarette smoking, health behaviors and risk factors found in other studies were confirmed in this Mexican American population. Few significant associations between smoking and health status were noted.

A new design of microwave interstitial applicators for hyperthermia with improved treatment volume.

New 915 MHz microwave interstitial applicators with improved treatment volume have been developed for clinical hyperthermia. The applicators are made from semi-rigid miniature coaxial cables by removing sections of the outer copper conductor to create multiple nodes while preserving the integrity of the teflon dielectric insulators. The homogeneity of the temperature distribution along the longitudinal axis is optimized by empirically adjusting the spacing of the gaps between sections of the outer conductor along the length of the applicator. In vitro and in vivo testing of the two-node and three-node microwave applicators show that the treatment volume can be improved by 100% over that of a one-node microwave applicator.

Growth, erythrocyte glutathione reductase and liver flavin as indicators of riboflavin status in turkey poults.

1. Turkey poults were fed on an isolated soya protein diet supplemented with 0, 2, 4 or 8 mg riboflavin/kg between 7 and 28 d of age. Maximum body weight was attained with the 4 and 8 mg diets, and poults fed on the supplemented diet did not survive until 21 d of age. The erythrocyte glutathione reductase activation coefficient (EGRAC) of the poults was inversely related to dietary riboflavin at all ages. At 28 d of age the values for EGRAC of poults fed on the 2, 4 and 8 mg diets were 2.25, 1.59 and 1.25 respectively; at 31 d, following a single oral dose of 10 mg riboflavin administered on day 28, the same poults had EGRAC values of 1.36, 1.17 and 1.26. 2. In a second experiment, poults were fed on diets supplemented with 0, 2, 4, 8 or 12 mg riboflavin/kg. Maximum body weight was attained with the 4, 8 and 12 mg diets. At 28 d of age the EGRAC values for the 0, 2, 4, 8 and 12 mg treatments were 2.36, 2.34, 1.59, 1.28 and 1.18 respectively. The concentration of flavin in the liver was not related to riboflavin intake, whereas total flavin in the liver was positively related, being on average 94, 206, 400, 440 and 413 micrograms flavin/liver for the 0, 2, 4, 8 and 12 mg treatments respectively. 3. It is concluded that EGRAC is a sensitive indicator of riboflavin status in the turkey poult and that an EGRAC of 1.6 indicates a marginal riboflavin status. It is also concluded that liver total flavin reflects merely the effect of riboflavin on growth and liver size.

Responses of laying hens to a low-protein diet supplemented with essential amino acids, L-glutamic acid and/or intact protein.

1. Three sequential experiments, each lasting 8 weeks, were carried out on 576 singly-caged light hybrids. 2. In experiment 1 egg production was 84% using a conventional control diet, 61% with a basal low-protein diet, and 79% with the basal diet supplemented with 10 essential amino acids+L-glutamic acid (GA). 3. In experiment 2 supplementation with lysine and methionine (L+M) alone increased egg production significantly from 54 to 72%, compared with 83% with the conventional diet. 4. In experiment 3 egg production was 55% with the basal diet, 71% with the basal diet+L+M, 75% with a diet containing 141 g protein/kg+L+M, and 73% with the conventional diet. 5. In all three experiments supplementation with GA alone either gave no significant response or a depression in production. 6. Daily intakes of 1-24 g nitrogen as non-essential amino acids and 13 to 14 g total crude protein per bird resulted in good egg production. Supplementation of the basal diet with L+M resulted in a daily intake of 413 mg methionine/bird day which was considered adequate, and a daily intake of 710 mg lysine which was considered slightly inadequate.