Potential alteration of tumor microenvironments by ß-mercaptoethanol.

The therapeutic effectiveness of immune checkpoint inhibitors in cancer patients is quite profound. However, it is generally accepted that further progress is curtailed by accompanying adverse events and by low cure rates linked to the tumor microenvironment. The multitudes of immune processes altered by low-molecular-weight thiols published over the past decades suggest they have potential to alter tumor microenvironment processes which could result in an increase in immune checkpoint inhibitor survival rates. Based on one of the most studied and most potent low-molecular-weight thiols, ß-mercaptoethanol (BME), it is proposed that clinical assessment be undertaken to identify any BME benefits with relevance for proliferation/differentiation of immune cells, lymphocyte exhaustion, immunogenicity of tumor antigens and inactivation of suppressor cells/factors. The BME alterations projected to be most effective are: maintenance/replacement of glutathione in lymphocytes via facilitation of cysteine uptake, inhibition of suppressor cells/soluble factors and inactivation of free-radical, reactive oxygen species.

A review: alteration of in vitro reproduction processes by thiols -emphasis on 2-mercaptoethanol.

Descriptions of organosulfurs altering biologically relevant cellular functions began some 40 years ago when murine in vitro cell mediated and humoral immune responses were shown to be dramatically enhanced by any of four xenobiotic, sulfhydryl compounds-2-mercaptoethanol (2ME), dithiothreitol (DTT), glutathione, and L-cysteine; the most effective were 2ME and DTT. These findings triggered a plethora of reports defining 2ME benefits for a multitude of immunological processes. This in turn led to investigations on 2ME alterations of (a) immune functions in other species, (b) activities of other cell-types, and (c) in vivo diseases. In addition, these early findings preceded the identification of previously undefined anticarcinogenic chemicals in specific foods as organosulfurs. Taken all together, there is little doubt that organosulfur compounds have enormous benefits for cellular functions and for a multitude of diseases. Issues of importance still to be resolved are (a) clarification of mechanisms that underlie alteration of in vitro and in vivo processes and perhaps more importantly, (b) which if any in vitro alterations are relevant for (i) alteration of in vivo diseases and (ii) identification of other diseases that might therapeutically benefit from organosulfurs. As one means to address these questions, reviews of different processes impacted by thiols could be informative. Therefore, the present review on alterations of in vitro fertilization processes by thiols (mainly 2ME, since cysteamine alterations have been reviewed) was undertaken. Alterations found to occur in medium supplemented with 2ME were enhancement, no effect, or inhibition. Parameters associated with which are discussed as they relate to postulated thiol mechanisms.

Alteration of radiation-sensitive processes associated with cancer and longevity by dietary 2-mercaptoethanol.

BACKGROUND: Previous results demonstrated dietary 2-mercaptoethanol (2-ME) delayed appearance of cancer in certain murine strains. In addition, it had a benefit not found with other organosulfurs, in that it completely prevented spontaneous development of cancer in BXSB-Yaa + over an entire lifespan. AIMS: These benefits raise the question: What, if any, alteration of radiation-induced tumorigenesis would 2-ME impart that may differ from that of other sulfur antioxidants? This is relevant based on the extensive use of radiation in diagnoses and therapy and 2-ME's superior in vitro and in situ immune enhancement properties. MATERIALS AND METHODS: This was addressed by exposing long-lived, B10.A (4R) mice to sublethal, 5.5 Gy ionizing gamma-rays and then tumor development monitored over a lifetime. STATISTICAL ANALYSIS: Two-tailed P-values were determined using the Fischer's Exact Test. RESULTS: The only tumors detected were mammary and only in animals that were both exposed to radiation and not treated with 2-ME. The 43% incidence differed significantly from the absence of tumors in non-irradiated mice that were or were not exposed to 2-ME and in those irradiated and treated daily with 2-ME, irrespective of whether treatment was started prior to or post irradiation. However, quite unexpectedly, radiation shortened longevity 29% from undefined causes, including cancer, in animals pretreated with 2-ME; longevity was not altered in those not pretreated or if treatment was started post-irradiation. CONCLUSIONS: The findings have relevance for cancer prevention and the controversy relative to ''long term survival/safety'' of currently used antioxidants as free radical scavengers in humans undergoing radiotherapy.

Review: 2-mercaptoethanol alteration of in vitro immune functions of species other than murine.

Descriptions that organosulfurs could alter biologically relevant cellular functions began some 40years ago when cell mediated and humoral murine in vitro immune responses were reported to be dramatically enhanced by any of four xenobiotic, sulfhydryl compounds-2-mercaptoethanol (2-ME), dithiothreitol, glutathione, and l-cysteine; the most effective of the four was 2-ME. These findings triggered a plethora of reports defining 2-ME benefits for a multitude of immunological processes, primarily with murine models. This led to investigations on 2-ME alterations of (a) immune functions in other species, (b) activities of other cell-types, and (c) in situ diseases. In addition, the early findings may have been instrumental in the identification of the previously undefined anticarcinogenic chemicals in specific foods as organosulfurs. Outside the plant organosulfurs, there are no comprehensive reviews of these areas to help define mechanisms by which organosulfurs function as well as identify potential alternative uses. Therefore, the present review will focus on 2-ME alterations of in vitro immune functions in species other than murine; namely, fish, amphibian, reptile, avian, whales, dolphins, rat, hamster, rabbit, guinea pig, feline, canine, porcine, ovine, bovine, and human. Processes, some unique to a given species, were in general, enhanced and in some cases dependent upon the presence of 2-ME. The largest benefits occurred in media that were serum free, followed by those in autologous serum and then fetal bovine serum supplemented medium. Concentrations of 2-ME were generally in the low µM range, with exceptions of those for salamander (20mM), turtles (70mM) and dolphins (7mM). The few studies designed to assess mechanisms found that changes induced by 2-ME were generally accompanied by alterations of reduced/oxidized glutathione cellular concentrations. The major benefit for most studies, however, was to increase the sensitivity of the culture environment, which permitted a specific process to be more easily dissected.

Dietary supplemented 2-mercaptoethanol prevents spontaneous and delays virally-induced murine mammary tumorigenesis.

There seems to be little doubt that organosulfur compounds have enormous benefits for biological processes, especially those of diseases like cancer. The preliminary results herein define a cancer model in which benefits/mechanisms of multitudes of xenobiotic and nature's organosulfurs could easily be compared. Mice from three strains with a high incidence for naturally occurring tumors were treated daily with 2-mercaptoethanol (2-Me) starting at weaning. The 100% tumor incidence of undefined etiology in untreated BXSB-Yaa (+) males was completely prevented by 2-Me. In contrast, 2-Me treatment of female and male C3H.OL and C3H.OH congenic strains, did not change the 100% tumor incidence due to milk-borne retrovirus, MMTV(S), but did: (1) delay the appearance of tumors by 42%; (2) increase longevity 56%; and (3) increase longevity, post-tumor detection, 95%. The addition of these results to the increasingly impressive anti-cancer benefits of simple xenobiotic organosulfurs raise the question: Can they be adapted for use as a preventive modality for human cancer?

Anticancer activity and chemoprevention of xenobiotic organosulfurs in preclinical model systems.

There seems to be little doubt that xenobiotic and plant derived organosulfur compounds have enormous benefits for in vitro cellular functions and for a multitude of diseases, including cancer. Since there are numerous reviews on anticancer activities of plant organosulfurs, the focus herein will be on alterations associated with xenobiotic organosulfurs. Benefits of 2-mercaptoethanol (2-Me), N-Acetyl-cysteine, cysteamine, thioproline, piroxicam, disulfiram, amifostine, sulindac, celecoxib, oltipraz and their derivates on transplanted homologous tumors and on autochthonous cancers with a viral-, radiation-, chemical carcinogen-, and undefined-etiology are assessed. Because all organosulfurs were not tested for activity in each of the etiology categories, comparative evaluations are restricted. In general, all 'appeared' to lower the incidence of cancer irrespective of etiology; however, since most of these values were determined at ages much younger than at a natural-end-of-life-age, differences most likely, instead, reflect a delayed initiation and/or a slowed progression of tumorigenesis. The poorest, long-term benefits of early intervention protocols occurred for viral- and chemical carcinogen-induced cancers. In addition, once tumorigenesis was beyond the initiation stage, outcomes of organosulfur therapies were extremely poor, indicating that they will not be of significant value as stand alone treatments. More importantly, except for the lifetime prevention of spontaneous and radiation-induced mammary tumors by daily dietary 2-Me, similar life long prevention of tumorigenesis was not achieved with other xenobiotics or any of nature's plant organosulfurs. These results raise an interesting question: Is the variability in incidence found for different organosulfurs associated with (a) their structure, (b) the length of the untreated latency period, (c) treatment duration/dose, and/or (d) the etiology-inducing agent?

Alteration of GI symptoms in a cow with Johne disease by the dietary organosulfur, 2-mercaptoethanol.

Sub-phenotypes of inflammatory bowel disease (IBD)-Crohn disease, ulcerative colitis and some cases of irritable bowel syndrome-are generally considered a consequence of gastrointestinal inflammation of unknown etiology. Conventional therapy and more recently biologic agents, all with varying degrees of drawbacks, have resulted in improved control of these diseases. However, as the incidence and prevalence continue to rise, needs for prevention, permanent remission and cures remain unmet, plus there still remain needs for improved control of symptoms, such as pain and diarrhea. The case report herein describes a serendipitous, novel means for curtailing these symptoms associated with a bovine gastrointestinal disease that may have applicability for patients with diseases characterized by abdominal-visceral pain and diarrhea.

A Potential 'Curative' Modality for Crohn's Disease---Modeled after Prophylaxis of Bovine Johne's Disease.

A naturally occurring, gastrointestinal disorder of ruminants (Johne's disease) is a chronic, debilitating, lethal disease. The causative agent is Mycobacterium avium subspecies paratuberculosis (MAP). Exposure that leads to disease occurs primarily in utero and/or during the neonatal period. Outside a dietzia probiotic treatment, there are no preventive/curative therapies. Interestingly, MAP is at the center of a controversy as to its role (cause of, perpetuate of, innocent bystander) in Crohn's disease, ulcerative colitis, irritable bowel syndrome, diabetes, sarcoidosis, Blau syndrome, and multiple sclerosis-diseases in which the incidence of systemic MAP is higher than that in the general population. Conventional therapeutic modalities, including biologic agents, for the majority of these diseases are, in general, directed at curtailing processes that are an intricate part of inflammation, with goals to induce and maintain remission. Most possess side effects of varying severity, lose therapeutic value, and more importantly, few are directed at prevention, attainment of long lasting remissions or cures, and essential none at reduction/elimination of MAP. This report presents a rationale for how/why Dietzia subsp. C79793-74 should be clinically evaluated for efficacy in patients with IBD. Arguments are based on previous studies that demonstrated (a) clinical similarities of Johne's disease and Crohn's disease, (b) inhibition of growth of MAP by Dietzia under specific culture conditions, (c) safe usage for extended daily treatments of adult cattle (up to 24 months), and (d) when used as a probiotic, curtailed diarrhea and cured 40% of adult cattle with early stage paratuberculosis.

A 60-day probiotic protocol with Dietzia subsp. C79793-74 prevents development of Johne's disease parameters after in utero and/or neonatal MAP infection.

The research reported herein was designed to assess whether the bacterium, Dietzia subspecies C79793-74, used as a probiotic, could prevent development of parameters indicative of bovine paratuberculosis after potential in utero, birthing and neonatal (colostrum) exposure to Mycobacterium avium subspecies paratuberculosis (MAP). Such exposure avenues are especially relevant for dairy farms practicing good management procedures since calves on these farms could be infected via dams that have yet to be identified as MAP-positive. Indeed, of 18 calves in the present study that became paratuberculosis parameter-positive, five had dams that were negative for all parameters pre-calving. Parameters used herein to define paratuberculosis status were serum ELISA, serum agar gel immunodiffusion, cultureable fecal MAP, histopathology at necropsy and clinical disease. Thirty-four newborn calves, whose dams were paratuberculosis-positive, were assigned to four different treatment groups. Ten were treated daily for 60 days with viable Dietzia added to their antibiotic-free milk feedings; none became positive for any parameter with age. In contrast, seven of eight calves that were not treated became positive for one or more paratuberculosis-associated parameter. Sixteen calves were treated with viable Dietzia for the first two days of life; eight were then not treated further, whereas the other eight were treated an additional 58 days with Dietzia added to tetracycline-fortified milk (Dietzia is sensitive to tetracycline). In these two groups, positivity developed in five of eight and six of eight, respectively. These results indicated that (a) a daily, 60-day treatment with viable Dietzia effectively prevented development of parameters indicative of paratuberculosis and (b) this treatment, in combination with good management practices, has the potential to eradicate MAP from animals/herds, which should curtail the spread of MAP. Such results should significantly reduce human exposure to MAP, which in turn, could have relevance for the controversial role of MAP in Crohn's disease, type-1 diabetes mellitus, sarcoidosis, Blau syndrome, ulcerative colitis, irritable bowel syndrome and multiple sclerosis.

Successful treatment of asymptomatic or clinically terminal bovine Mycobacterium avium subspecies paratuberculosis infection (Johne's disease) with the bacterium Dietzia used as a probiotic alone or in combination with dexamethasone: Adaption to chronic human diarrheal diseases.

A naturally occurring gastrointestinal disease, primarily of ruminants (Johne disease), is a chronic debilitating disease that is caused by Mycobacterium avium subspecies paratuberculosis (MAP). MAP infection occurs primarily in utero and in newborns. Outside our Dietzia probiotic treatment, there are no preventive/curative therapies for bovine paratuberculosis. Interestingly, MAP is at the center of controversy as to its role in (cause of) Crohn disease (CD) and more recently, its role in diabetes, ulcerative colitis, and irritable bowel syndrome (IBS); the latter two, like CD, are considered to be a result of chronic intestinal inflammation. Treatments, both conventional and biologic agents, which induce and maintain remission are directed at curtailing processes that are an intricate part of inflammation. Most possess side effects of varying severity, lose therapeutic value, and more importantly, none routinely result in prevention and/or cures. Based on (a) similarities of Johne disease and Crohn disease, (b) a report that Dietzia inhibited growth of MAP under specific culture conditions, and (c) findings that Dietzia when used as a probiotic, (i) was therapeutic for adult bovine paratuberculosis, and (ii) prevented development of disease in MAP-infected calves, the goal of the present investigations was to design protocols that have applicability for IBD patients. Dietzia was found safe for cattle of all ages and for normal and immunodeficient mice. The results strongly warrant clinical evaluation as a probiotic, in combination with/without dexamethasone.

Comparison of ante-mortem assays to assess progression/regression of paratuberculosis in individual dairy animals.

Johne's disease, caused by Mycobacterium avium, subspecies paratuberculosis (MAP) is becoming increasingly widespread on dairy farms worldwide, due in part, to the absence of vaccine/drug or curative modalities.  This spread is of concern since MAP is at the center of a controversy as to its role in Crohn's disease.  None of the methods presently available to define paratuberculosis in cattle have been examined for their ability to assess progression/regression of any treatment or intervention of this disease   The research presented herein, therefore was designed to assess the reliability and accuracy of available ante-mortem assays to predict disease change of individual animals undergoing a probiotic, potentially therapeutic, treatment.  Paratuberculosis positive (n = 75) and negative (n = 10) animals were longitudinally monitored over their natural lifetimes with specific serum antibody and fecal shedding assays, and for development of end-stage clinical disease.  Longitudinal, increasing/decreasing serum ELISA values were associated with, and predictive of, progression/regression of disease.  Changes in fecal shedding and serum AGID were of value at only specific stages.  Documentation that ELISA-positive animals were positive for paratuberculosis was done by a compilation of ELISA-independent assays--succumbing with end-stage clinical disease, autopsy, AGID, and MAP fecal shedding.

Assessment of Dietzia subsp. C79793-74 for treatment of cattle with evidence of paratuberculosis.

The objective of the present investigation was to determine whether the bacterium Dietzia subsp. C79793-74, previously shown to inhibit growth of Mycobacterium subsp. paratuberculosis under in vitro culture conditions, has therapeutic value as a probiotic for adult cattle with paratuberculosis.  Animals were obtained from several herds with evidence of disease based on seropositivity and/or fecal shedding.  Sixty-eight cows with initial evidence of Stage II or III paratuberculosis and 2 with an initial Stage IV disease were evaluated longitudinally.  Animals were either treated daily with variable, disease-dependent doses of Dietzia (n = 48) or left untreated (n = 22).  Clinical aspects of disease (diarrhea, emaciated, cachectic and appetite) were recorded until the animal recovered or required euthanasia due to advanced clinical paratuberculosis or other severe conditions.  Paratuberculosis parameters-antibody serology (ELISA, AGID) and fecal culture-were longitudinally monitored over the lifetime of each animal.  The results indicated that daily treatment with Dietzia was therapeutic for paratuberculosis cows based on: (a) longitudinal decline in ELISA values only occurred in animals that were treated; (b) prolonged survival was dependant upon treatment--the length being directly associated with low initial ELISA values; and (c) treated animals were the only ones cured of disease.  Further investigations are envisaged to determine optimal, long-term dosages that may result in even better therapeutic outcomes as well as to evaluate potential application for therapy of the Johne's disease, human-counterpart, Crohn's disease.

Obesity, longevity, quality of life: alteration by dietary 2-mercaptoethanol.

Previous investigations demonstrated that optimization of murine immunological reactivity in tissue culture required a sulfhydryl compound; the most effective being 2-mercaptoethanol (2-Me). Since these reports, 2-Me was found beneficial for both growth/function of other cell-types in vitro, including those of other species, and when fed orally, it impeded and/or reversed some in situ physiological changes associated with aging. More recently, thiol-containing compounds possessing oxidation-reduction potentials weaker than 2-Me were found to impart beneficial effects for many other, including human, diseases. Based on these effects, the research herein addressed the question: What consequences might dietary 2-Me impart on health and disease of mice other than those associated with aging? The main parameters monitored over the lifetime of individual animals exposed to dietary 10⁻³ M 2-Me in their drinking water were: quality of life (obesity and development of recumbent, emaciated and/or cachectic health); longevity; and appearance of tumors. Instead of anticipated toxic attributes, the following unique benefits were found; mean survival of a moderately-lived strain (A/J) was increased 40.8%, high-fat-diet obesity was curtailed in C57BL/10 mice, and a goal of aging intervention protocols, namely preventing loss of quality of life during aging (recumbent, emaciated and/or cachectic) was achieved. Various mechanisms are discussed as they pertain to these findings.

Longevity of SLE-prone mice increased by dietary 2-mercaptoethanol via a mechanism imprinted within the first 28 days of life.

In the preceding report, moderately lived-mice fed dietary 2-mercaptoethanol (2-Me) had their life extended, whereas long-lived mice were found to have the quality of life improved, but not extended, and did not develop high fat-diet obesity. In the present report, alteration of longevity of mice prone to develop spontaneous, systemic lupus erythematosus (SLE) by dietary 2-Me was determined. NZB, NZW, (NZW x NZB) F1-hybrid, BXSB/MpJ, BXSB-Yaa+/J, MRL/MpJ and MRL/MpJ-Faslpr mice received drinking water, without or with 2-Me at concentrations of 10⁻³ or 10⁻² M. Therapeutic benefit was assessed by changes in longevity. The median survival of MRL/MpJ males was increased from 443 to 615 days and those of (NZW x NZB) F1 and NZB males and females were increased approximately 2-fold. The most unexpected finding was that longevity of F1 males was significantly extended irrespective of whether dietary exposure to 2-Me was initiated at 28 days of age, at 50 days of age, or initiated during gestation (and then terminated at weaning--28 days of age). Survival of F1-hybrids in which treatment was initiated in utero or at 28 days of age was not significantly different, whereas if initiation was delayed until 50 days of age, survival was >200 days shorter. Survival of male MRL/MpJ-Fas lpr and BXSB/MpJ (Yaa-), two strains with genetically controlled accelerated SLE, was not altered by 2-Me when started at 50 days. Various alternatives are discussed regarding potential long-lasting mechanisms imprinted early in life. Even though present day treatments of rodent SLE are generally aimed at controlling specific immunological events, with or without survival benefits, or are procedures presently unsuitable for therapeutic use in humans, the findings presented herein seem worthy of clinical evaluation.