Immune activation of Bio-Germanium in a randomized, double-blind, placebo-controlled clinical trial with 130 human subjects: Therapeutic opportunities from new insights.

REGISTERED CLINICAL TRIAL: [NCT03677921]; www.clinicaltrials.gov [KCT0002726]; https://cris.nih.go.kr.

Effects of germanium biotite supplement on immune responses of vaccinated mini-pigs to foot-and-mouth disease virus challenge.

Since the outbreaks of foot-and-mouth disease (FMD) in South Korea in 2010-2011, a trivalent vaccine has been used as a routine vaccination. Despite the high efficacy of the trivalent vaccine, low antibody formation was reported in the pig industry and there is considerable concern about the ability of the vaccine to protect against the Andong strain responsible for recent outbreaks in South Korea. To overcome these problems, immunostimulators have been widely used to improve vaccine efficacy in South Korea, although without any scientific evidence. Based on the current situation, the aim of this study was to investigate the effects of germanium biotite, a feed supplement used to enhance the immune system, on the immune responses to FMD vaccination through the Andong strain challenge experiment in trivalent vaccinated pigs. Following the challenge, the germanium biotite-fed pigs showed high levels of IL-8 in serum, and increased cellular immune responses to stimulation with the Andong strain antigen compared to nonsupplemented pigs. In addition, higher FMD virus (FMDV) neutralizing antibody titers were detected in the germanium biotite-fed group than in the nonsupplemented group before the challenge. The findings of this study indicate that germanium biotite supplement might enhance immune responses to the FMD vaccine in pigs.

Nanometer-long Ge-imogolite nanotubes cause sustained lung inflammation and fibrosis in rats.

BACKGROUND: Ge-imogolites are short aluminogermanate tubular nanomaterials with attractive prospected industrial applications. In view of their nano-scale dimensions and high aspect ratio, they should be examined for their potential to cause respiratory toxicity. Here, we evaluated the respiratory biopersistence and lung toxicity of 2 samples of nanometer-long Ge-imogolites. METHODS: Rats were intra-tracheally instilled with single wall (SW, 70 nm length) or double wall (DW, 62 nm length) Ge-imogolites (0.02-2 mg/rat), as well as with crocidolite and the hard metal particles WC-Co, as positive controls. The biopersistence of Ge-imogolites and their localization in the lung were assessed by ICP-MS, X-ray fluorescence, absorption spectroscopy and computed micro-tomography. Acute inflammation and genotoxicity (micronuclei in isolated type II pneumocytes) was assessed 3 d post-exposure; chronic inflammation and fibrosis after 2 m. RESULTS: Cytotoxic and inflammatory responses were shown in bronchoalveolar lavage 3 d after instillation with Ge-imogolites. Sixty days after exposure, a persistent dose-dependent inflammation was still observed. Total lung collagen, reflected by hydroxyproline lung content, was increased after SW and DW Ge-imogolites. Histology revealed lung fibre reorganization and accumulation in granulomas with epithelioid cells and foamy macrophages and thickening of the alveolar walls. Overall, the inflammatory and fibrotic responses induced by SW and DW Ge-imogolites were more severe (on a mass dose basis) than those induced by crocidolite. A persistent fraction of Ge-imogolites (15% of initial dose) was mostly detected as intact structures in rat lungs 2 m after instillation and was localized in fibrotic alveolar areas. In vivo induction of micronuclei was significantly increased 3 d after SW and DW Ge-imogolite instillation at non-inflammatory doses, indicating the contribution of primary genotoxicity. CONCLUSIONS: We showed that nm-long Ge-imogolites persist in the lung and promote genotoxicity, sustained inflammation and fibrosis, indicating that short high aspect ratio nanomaterials should not be considered as innocuous materials. Our data also suggest that Ge-imogolite structure and external surface determine their toxic activity.

Supplementation of dietary germanium biotite enhances induction of the immune responses by foot-and-mouth disease vaccine in cattle.

BACKGROUND: After the recent outbreak of foot-and-mouth disease (FMD) in Korea, a vaccination policy has been applied to control the disease. In addition, several non-specific immune stimulators have been used without any scientific evidence that they would enhance the immune response after FMD vaccination and/or protect against FMD. Based on the current situation, the aim of this study was to evaluate the effect of the non-specific immune stimulator germanium biotite on FMD vaccination and immune responses in cattle. To achieve our goal, immune responses to FMD vaccination, such as levels of IgG and IgA, antibody duration, and virus-neutralizing titers were investigated after germanium biotite feeding. The PBMC typing and proliferative response after stimulation with mitogens, the cytokines expression level of PBMC, and the lysozyme activity in the serum were measured to evaluate the immune enhancing effects of germanium biotite following its administration. RESULTS: Following the first vaccination, high level of IgG (at 4 weeks) and IgA (at 2 and 31 weeks) titers in serum and saliva were observed in the germanium biotite-feeding group (p < 0.05). The germanium biotite group also showed high and longstanding inhibition percentage value in ELISA assay at 31 weeks (p < 0.05). Generally, higher virus-neutralizing antibody titers were observed in the feeding group at 20 and 31 weeks after vaccination. Following the feeding germanium biotite, the germanium biotite group showed increased subpopulation of CD4+ lymphocytes and MHC I+II+ cells in PBMCs at 23 week, responding to stimulation of ConA. The levels of IFN-γ (at 3 and 8 weeks), IL-1α (at 3, 11, and 23 weeks), IL-1ß (at 3, 8, and 11 weeks), and IL-4 (at 8 and 11 weeks) gene expression were also significantly increased in the feeding group (p < 0.01 and p < 0.05). Feeding with germanium biotite increased the lymphocytes' proliferative response to the stimulation of ConA and LPS at 23 weeks and lysozyme activity at 9 weeks after feeding. CONCLUSIONS: These results suggest that germanium biotite feeding could increase the protection against FMD virus infection via the induction of higher humoral and cellular immune responses in cattle.

The Oral Intake of Organic Germanium, Ge-132, Elevates α-Tocopherol Levels in the Plas-ma and Modulates Hepatic Gene Expression Profiles to Promote Immune Activation in Mice.

The common water-soluble organic germanium compound poly-trans-[(2-carboxyethyl) germasesquioxane] (Ge-132) exhibits activities related to immune responses and antioxidant induction. In this study, we evaluated the antioxidative effect of dietary Ge-132 in the plasma of mice. Male ICR mice (seven mice per group) received an AIN-76 diet with 0.05% Ge-132; three groups received the Ge-132-containing diet for 0, 1 or 4 days. The plasma alpha-tocopherol (α-tocopherol) concentration increased from 6.85 to 9.60 µg/ml after 4 days of Ge-132 intake (p<0.05). We evaluated the changes in hepatic gene expression related to antioxidative activity as well as in the entire expression profile after one day of Ge-132 intake, using DNA microarray technology. We identified 1,220 genes with altered expression levels greater than 1.5-fold (increased or decreased) as a result of Ge-132 intake, and α-tocopherol transfer protein (Ttpa) gene expression was increased 1.62-fold. Immune activation was identified as the category with the most changes (containing 60 Gene Ontology (GO) term biological processes (BPs), 41 genes) via functional clustering analysis of altered gene expression. Ge-132 affected genes in clusters related to ATP production (22 GO term BPs, 21 genes), lipid metabolism (4 GO term BPs, 38 genes) and apoptosis (5 GO term BPs). Many GO term BPs containing these categories were significantly affected by the Ge-132 intake. Oral Ge-132 intake may therefore have increased plasma α-tocopherol levels by up-regulating α-tocopherol transfer protein (Ttpa) gene expression.

Antiviral effect of dietary germanium biotite supplementation in pigs experimentally infected with porcine reproductive and respiratory syndrome virus.

Germanium biotite (GB) is an aluminosilicate mineral containing 36 ppm germanium. The present study was conducted to better understand the effects of GB on immune responses in a mouse model, and to demonstrate the clearance effects of this mineral against Porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected pigs as an initial step towards the development of a feed supplement that would promote immune activity and help prevent diseases. In the mouse model, dietary supplementation with GB enhanced concanavalin A (ConA)-induced lymphocyte proliferation and increased the percentage of CD3+CD8+ T lymphocytes. In pigs experimentally infected with PRRSV, viral titers in lungs and lymphoid tissues from the GB-fed group were significantly decreased compared to those of the control group 12 days post-infection. Corresponding histopathological analyses demonstrated that GB-fed pigs displayed less severe pathological changes associated with PRRSV infection compared to the control group, indicating that GB promotes PRRSV clearance. These antiviral effects in pigs may be related to the ability of GB to increase CD3+CD8+ T lymphocyte production observed in the mice. Hence, this mineral may be an effective feed supplement for increasing immune activity and preventing disease.

Antiviral effect of dietary germanium biotite supplementation in pigs experimentally infected with porcine reproductive and respiratory syndrome virus

Germanium biotite (GB) is an aluminosilicate mineral containing 36 ppm germanium. The present study was conducted to better understand the effects of GB on immune responses in a mouse model, and to demonstrate the clearance effects of this mineral against Porcine reproductive and respiratory syndrome virus (PRRSV) in experimentally infected pigs as an initial step towards the development of a feed supplement that would promote immune activity and help prevent diseases. In the mouse model, dietary supplementation with GB enhanced concanavalin A (ConA)-induced lymphocyte proliferation and increased the percentage of CD3+CD8+ T lymphocytes. In pigs experimentally infected with PRRSV, viral titers in lungs and lymphoid tissues from the GB-fed group were significantly decreased compared to those of the control group 12 days post-infection. Corresponding histopathological analyses demonstrated that GB-fed pigs displayed less severe pathological changes associated with PRRSV infection compared to the control group, indicating that GB promotes PRRSV clearance. These antiviral effects in pigs may be related to the ability of GB to increase CD3+CD8+ T lymphocyte production observed in the mice. Hence, this mineral may be an effective feed supplement for increasing immune activity and preventing disease.

Dermal absorption of inorganic germanium in rats.

So-called germanium 'health' products including dietary supplements, cosmetics, accessories, and warm bath service containing germanium compounds and metalloid are popular in Japan. Subchronic and chronic oral exposure of germanium dioxide (GeO(2)), popular chemical form of inorganic germanium causes severe germanium toxicosis including death and kidney dysfunction in humans and experimental animals. Intestinal absorption of neutralized GeO(2) or germanate is almost complete in humans and animals. However, it is not known whether germanium is cutaneously absorbed. We tested dermal absorption of neutralized GeO(2) or germanate using male F344/N rats. Three groups of rats were treated with a 3-h topical application of hydrophilic ointment containing graded level of neutralized GeO(2) (pH 7.4): 0, 0.21 and 0.42 mg GeO(2)/g. Germanium concentration in blood and tissues sampled from rats after topical application of inorganic germanium was measured by inductively coupled plasma-mass spectrometry. Animals topically applied 0.42 mg GeO(2)/g ointment had significantly higher germanium concentrations in plasma, liver, and kidney than those of rats that received no topical germanium. The results indicate that skin is permeable to inorganic germanium ion or germanate and recurrent exposure of germanium compounds may pose a potential health hazard.

Effect of germanium, poly-trans-[2-carboxyethyl] germasesquioxane on natural killer (NK) activity in dogs.

In an attempt to study the immunological effects on normal and adenocarcinoma (AC) dogs, the natural killer (NK) activity was determined. Augmentation of NK cell activities in responsive normal and AC dogs that indicatedanergy in the phaseolus vulgaris agglutinin (PHA) skin-test was manifested when the animals were accordingly treated with germanium (GN).

Complete remission of pulmonary spindle cell carcinoma after treatment with oral germanium sesquioxide.

Spindle cell carcinoma (SCC) is a rare form of lung cancer representing 0.2 to 0.3% of all primary pulmonary malignancies. Even with combined surgery, chemotherapy, and radiation therapy, these tumors are associated with a poor prognosis and only 10% of patients survive 2 years after diagnosis. We describe a patient with an unresectable SCC who, following no response to conventional treatment with combined modality therapy, chose to medicate herself with daily doses of germanium obtained in a health food store. She noted prompt symptomatic improvement and remains clinically and radiographically free of disease 42 months after starting her alternative therapy.

Effects of germanium oxide and other chemical compounds on phenylmercury acetate-induced genotoxicity in cultured human lymphocytes.

Phenylmercury acetate (PMA), which not only causes an elevation of sister chromatid exchanges (SCEs) but also induces high frequency of endoreduplication in human lymphocytes, may be genotoxic to humans. The major aim of our study was to investigate the effects of germanium oxide (GeO2), D-penicillamine (D-PA), dimercaprol (BAL), and diltiazem (DTM) on PMA-induced genotoxicity as quantified by SCEs. All concentrations of the four chemical compounds tested alone did not induce genotoxicity in cultured human lymphocytes. However, GeO2 significantly inhibited PMA-induced genotoxicity in a concentration-dependent manner. Similarly, D-PA at concentrations of 3 microM and 10 microM, and BAL at a concentration of 30 microM produced the antigenotoxic effects. In addition, GeO2 (1.5 microM) significantly reversed an increase of endoreduplication frequency caused by PMA. In a cell cycle kinetic study, GeO2 (0.5-5.0 microM) reversed the inhibition of PMA on the proliferating rate index (PRI) of lymphocytes. On the contrary, both D-PA and DTM at concentrations of 30-300 microM markedly potentiated PMA-induced inhibition of PRI. These findings show that GeO2, D-PA and BAL could antagonize PMA-induced genotoxicity, and GeO2 appears to be the most effective.

Effect of pretreatment of germanium-132 on Na(+)-K(+)-ATPase and galactose cataracts.

PURPOSE: Recently, we reported that topical administration of 2-carboxyethyl germanium sesquioxide (Ge-132) concurrently with 50% galactose feeding delayed the establishment of mature cataracts and reduced advance glycation product. This study was to determine the effect of pretreatment of Ge-132 on galactose associated morphological changes and Na(+)-K(+)-ATPase activity. METHODS: Young Sprague Dawley rats received topical eye drops four times a day of either saline or Ge-132 seven days prior to the 50% galactose diet and during galactose feeding. At desired intervals the lenses were extracted, photographed and processed for either light microscopy, scanning electron microscopy or the determination of Na(+)-K(+)-ATPase activity. RESULTS: In Ge-132 pretreated lenses as compared to saline pretreated lenses the following results were observed: (a) the galactose-induced morphological alterations in the majority of lenses were delayed and (b) Na(+)-K(+)-ATPase activity was protected. CONCLUSIONS: Our previous and current studies show that in addition to osmotic stress post-translational protein modification, such as glycation, including enzymes may play a role in initiating changes that lead to cataract development. The inhibition of protein glycation by antiglycating compounds, such as Ge-132, delays sugar cataract formation. Currently, we are investigating the status of protein glycation and advanced glycation end products following pretreatment with Ge-132 and the role of Ge-132 on the activities of enzymes such as aldose reductase and Na(+)-K(+)-ATPase.

Pharmacokinetics of germanium after po beta-carboxyethylgermanium sesquioxide in 24 Chinese volunteers.

AIM: To compare the pharmacokinetics after po different doses of beta-carboxyethylgermanium sesquioxide (Ge-132). METHODS: An atomic absorption spectrophotometric system was used to measure germanium concentrations in plasma and urine samples after po Ge-132 1 (low dose, LD), 2.5 (medium dose, MD), and 4 (high dose, HD) g.m-2 in 24 healthy volunteers (one dose per 8 subjects). RESULTS: T1/2 alpha (LD, 1.2 +/- 0.7 h; MD, 1.1 +/- 0.6 h; HD, 1.2 +/- 0.5 h), T1/2 beta (LD, 5.2 +/- 1.2 h; MD, 5.8 +/- 2.5 h; HD, 5.5 +/- 1.4 h) and Cl/F (LD, 33 +/- 12 L.h-1; MD, 35 +/- 10 L.h-1; HD, 33 +/- 11 L.h-1) were not dose-related. Tmax was between 0.75 h and 2 h. Cmax (LD, 5.3 +/- 2.2 mg.L-1; MD, 13 +/- 5 mg.L-1; HD 18 +/- 8 mg.L-1, HD) and AUC (LD, 31 +/- 13 mg.h.L-1; MD, 60 +/- 16 mg.h.L-1; HD, 79 +/- 42 mg.h.L-1) were positive correlation to the dose of Ge-132. Urine-eliminated germanium within 24 h accounted for 11 +/- 3% of LD, 9 +/- 3% of MD, and 6 +/- 5% of HD (calculated from Ge/F) and showed a negative correlation to the dose. CONCLUSION: 1) Intracorporal process of Ge after po Ge-132 coincided with the first-order absorption and elimination with two-compartment kinetic model; 2) The amount of germanium eliminated in urine was below 11%.

Acute and subacute inhalation toxicity of germanium dioxide in rats.

Two acute (4 hr) and one subacute (4 wk) inhalation toxicity studies on germanium dioxide (purity > or = 99%, mean particle size 1.7-2.6 microns) were conducted in young adult Wistar rats. In the acute studies, exposure of two groups of five rats of each sex to maximum attainable concentrations of either 3.10 g amorphous or 1.42 g hexagonal germanium dioxide/m3 for 4 hr was not lethal. In the subacute study, four groups of five rats of each sex were exposed to 0, 16, 72 and 309 mg hexagonal germanium dioxide/m3 for 6 hr/day, 5 days/wk during 4 wk. Two additional groups of 5 rats per sex, exposed either to 0 or to 309 mg/m3, were kept for a 33-day post-exposure period. At the end of the treatment period, changes were observed only in rats of the high concentration group: these changes were decreased body weight gain (both sexes), decreases in haematocrit (females) and thrombocyte count (both sexes), and increases in neutrophil count (both sexes) and white blood cell count (females). On clinical chemistry evaluation, decreased fasting blood glucose (females), decreased total protein concentration (both sexes), increased plasma alanine aminotransferase and aspartate aminotransferase activities (females), increased plasma urea nitrogen (males) and increased plasma bilirubin level (females) were observed. In addition, urinary volume was elevated, and urine density and pH were lowered in both sexes. Relative weights of kidneys, spleen, heart and lungs were higher than in controls. Microscopic examination revealed effects on renal tubular epithelium. Effects on growth, kidneys, and liver were still present at the end of the 33-day recovery period. It was concluded that the 4-hr LC50 value of amorphous germanium dioxide was greater than 3.10 g/m3 and that of the hexagonal form greater than 1.42 g/m3. The no-adverse-effect-level in the 4-wk study using hexagonal germanium dioxide was 72 mg/m3.

Effects of germanium and silicon on bone mineralization.

The chemical properties of Ge are similar to Si. This study investigated whether Ge can substitute for, or is antagonistic to, Si in bone formation. Sixty male weanling Sprague-Dawley rats were randomly assigned to treatment groups of 12 and 6 in a 2 x 4 factorially arranged experiment. The independent variables were, per gram fresh diet, Si (as sodium metasilicate) at 0 or 25 micrograms and Ge (as sodium germanate) at 0, 5, 30, or 60 micrograms. Results confirmed that Ge does not enhance Si deprivation and provided evidence that Ge apparently can replace Si in functions that influence bone composition. When Si was lacking in the diet, calcium and magnesium concentrations of the femur were decreased; this was reversed by feeding either Ge and/or Si. Similar effects were found for zinc, sodium, iron, manganese, and potassium of vertebra. There were some responses to Si deprivation that Ge could not reverse; Ge did not increase femur copper, sodium, or phosphorus or decrease molybdenum of vertebra, effects that were evoked by Si supplementation. Additionally, some findings suggested that 60 micrograms Ge/g diet could be a toxic intake for the rat. On the other hand, some responses induced by Ge indicate that this element may be acting physiologically other than as a substitute for Si. Germanium itself affected bone composition. Germanium supplementation decreased Si and molybdenum in the femur and increased DNA in tibia. Regardless of the amount of Si fed, animals fed 30 micrograms Ge/g diet had increased tibial DNA compared to animals fed 0 or 60 micrograms Ge; however, tibial DNA of animals fed 30 micrograms Ge was not statistically different from those animals fed 5 micrograms Ge. Thus, Ge may be of nutritional importance.

Multidisciplinary treatment of head and neck cancer using BCG, OK-432, and GE-132 as biologic response modifiers.

Since 1979, we have performed multidisciplinary treatment using intensive immunotherapy with biologic response modifiers (BRM) in combination with surgical treatment of oral cancer. Chemotherapy and radiotherapy were also included as part of the therapy. A historic control study was performed. Adjuvant therapy was administered by standardized methods, and the distribution of patients at various stages was similar between groups. The immunotherapy group showed a shorter treatment period, lower rates of recurrence, metastases, and side effects, greater histologic effects at the end of the first treatment, and a higher survival rate than the nonimmunotherapy group. Immunologically, immunotherapy tended to promote positive immune reactions and inhibit negative immune reactions.

Tubulointerstitial nephropathy persisting 20 months after discontinuation of chronic intake of germanium lactate citrate.

Two young human immunodeficiency virus (HIV)-infected patients, a 25-year-old woman and a 26-year-old man, consumed large amounts of germanium lactate citrate 18% as an "immunostimulant" for 9 months. The woman, who had stage II HIV infection, developed severe renal dysfunction (creatinine clearance, 7 mL/min/1.73 m2) and slight proteinuria (0.28 g/d) after ingesting 260 g germanium lactate citrate 18%. Hepatomegaly with liver dysfunction (SGOT, 102 U/L; gamma-glutamyl transferase (GT), 159 U/L) and lactic acidosis (plasma lactate, 7.3 mmol/L) developed simultaneously. Renal biopsy revealed tubulointerstitial nephropathy with vacuolar cell degeneration and periodic acid-Schiff-positive intracellular deposits mainly in distal tubules. Liver biopsy disclosed severe hepatic steatosis; liver function tests returned to normal within 5 weeks. Since renal failure persisted for 2 years after ingestion of germanium (creatinine clearance, 14 mL/min/1.73 m2; proteinuria, 0.84 g/d), a second renal biopsy was performed, which showed marked but focal distal tubular atrophy and slight interstitial fibrosis. The male patient, who had stage III HIV infection, had ingested the same compound; he presented with a creatinine clearance of 43 mL/min/m2 and proteinuria of 0.36 g/d. Renal biopsy disclosed tubulointerstitial changes similar to those found in the female patient. After 9 months off germanium, creatinine clearance remained unchanged. Neutron activation analysis of all biopsy specimens in both cases documented germanium concentrations 10 to 70 times normal in renal tissue and 140 times normal in liver tissue.

Nephrotoxicity of germanium compounds: report of a case and review of the literature.

A 55-year-old woman was admitted to our hospital, complaining of general malaise, muscular weakness, anorexia and weight loss. She had a history of ingesting of a certain germanium (Ge) compound over the preceding 19 months, with a total dose of 47 g as Ge element. She was found to have renal failure (blood urea nitrogen, 44 mg/dl; serum creatinine, 2.6 mg/dl) without abnormal findings in urinalysis, and muscular and nervous damage. Initially, polymyositis was diagnosed and prednisolone administered. However, no improvement was seen, and neuromuscular symptoms and signs steadily worsened, ending in death. Microscopic study of the kidney showed that lipofuscin granules increased in the cells of the thick ascending limb of Henle's loop to the distal convoluted tubule accompanying mild tubular atrophy and that some of the tubules of these segments had vacuolar degeneration or desquamation. No apparent glomerular and vascular changes were observed. High Ge content was found in serum, urine and various tissues, e.g., spleen, liver, kidney, adrenal gland and myocardium, while in controls Ge could not be detected in sera, urine or tissues. We also review case reports about Ge toxicity, and discuss the pathogenesis of renal failure induced by Ge compounds.

[Toxic damage of kidney, liver and muscle attributed to the administration of germanium-lactate-citrate]. / Toxische beschadiging van nier, lever en spier toegeschreven aan het gebruik van germanium-lactaat-citraat.

The case history is described of a woman aged 57 years with renal, hepatic and muscular damage attributed to intake of germanium lactate-citrate (a cumulative dose of 32.1 g germanium) over at least one year, as alternative treatment of metastatic breast cancer. Histological examination of biopsies showed highly vacuolated cytoplasm of the epithelial cells of the distal renal tubules and micro- and macrovesicular steatosis of centrilobular hepatocytes. After discontinuation of the germanium, serum aminotransferases and creatine kinase values returned to normal, but moderately severe renal impairment persisted.