Towards an understanding of biological role of colostrinin peptides.

The aim of this study was to elucidate the structure and possible function of colostrinin, also known as a proline rich polypeptide (PRP). The molecular weight of colostrinin was originally determined by gel filtration to be 17,200 daltons. In the presence of guanidinum chloride, however, the molecular weight was found to be about 6,000 daltons. Further studies utilizing high-performance liquid chromatography (HPLC) and mass spectroscopy revealed that colostrinin is a complex consisting of many low molecular-weight polypeptides. A total of 32 peptides were isolated from the original colostrinin preparation by HPLC and subjected to the N-terminal sequence analysis. The results of sequence analysis revealed significant homology of the peptides to three protein precursors: annexin, beta-casein, and a hypothetical beta-casein homolog. In addition, the sequence of several peptides showed no significant homology to any specific protein in the current GenBank database. The synthetic peptides of various lengths representing the N-terminal sequence of the colostrinin peptides were made to study some biological effects. Here we report that colostrinin and some of its component peptides are potent inducers of leukocyte proliferation and of certain cytokines. Also, a series of monospecific antibodies were produced in rabbits against the synthetic peptides. The antibodies were used to study the kinetic of antigen reduction in colostrum and mature milk following lambing. A threefold decrease was common for most antigens studied over the period of 72 h. Based on the results of these studies we postulate that colostrinin represents a diverse group of peptides produced in the mammary gland of mammals for the development of the optimal physiologic responses in offspring. Also, it is hoped that the beneficial use of colostrinin in Alzheimer's Disease (AD), recently reported elsewhere, will revive interest in its clinical application for treatment and/or prophylaxis of many age-related disorders.

Colostrinin: a proline-rich polypeptide (PRP) complex isolated from ovine colostrum for treatment of Alzheimer's disease. A double-blind, placebo-controlled study.

A proline-rich polypeptide (PRP) complex, subsequently called Colostrinin, was isolated from ovine colostrum. The complex showed immunomodulatory properties in mice, rats, and chickens, inducing maturation and differentiation of thymocytes. It was recently found that Colostrinin is a cytokine-like factor that acts as an inducer of interferon gamma (IFN-gamma) and other cytokines in human peripheral blood and cord blood leukocyte cultures and has psycho-immuno-enhancing activity in volunteers. These observations prompted us to study the effect of Colostrinin on patients with Alzheimer's disease (AD). Forty six AD patients were divided into 3 groups and randomly assigned to receive orally either Colostrinin (100 microg per tablet, every second day), commercially available bioorganic selenium (100 microg selenium per tablet, every second day) or placebo tablets. One cycle of the treatment lasted 3 weeks and was separated from the next cycle by a 2 week hiatus. Each patient received 10 cycles of treatment during the year of the clinical trial. Outcomes were assessed by psychiatrists blinded to the treatment assignment. Eight of the 15 AD patients treated with Colostrinin improved and in the 7 others the disease had stabilized. In contrast, none of the 31 patients from the selenium or placebo groups with similar mild or moderate AD improved. The administration of selenium promoted stabilization in 13 of the 15 patients, whereas in the placebo group only 8 of the 16 patients were stabilized at the 12 month trials end-evaluation. Colostrinin was found to be a remarkably safe drug. Mild and transient effects were anxiety, stimulation, insomnia, and tiredness. The results obtained showed that oral administration of Colostrinin improves the outcome of AD patients with mild to moderate dementia. The results are very encouraging and deserve further research.

Tumor-associated antigens are cytokine inducers and hyporeactivity factors to the immune system.

We investigated possible mechanisms leading to the inhibition of the immune system in people with chronic disorders. Tumor cell produce protein released into the circulation, such as tumor associated antigens, may play an important role in processes preceding paralysis of the immune system. To test this hypothesis the following tumor associated antigens were used: AFP, OFP, CA-125, CA-50 and CA-19-9. Their role was assessed by modulating cytokine production in cord blood lymphocytes and peripheral white blood cells obtained from grown population of patients treated with colostrinin, an cytokine inducer. PHA, LPS and colostrinin were used as positive control in those essays. Each antigen tested individually induced IFN, TNF alpha and IL-6 in dose dependent fashion. None of the tested cytokines were spontaneously released by the cells. Data generated from these experiments indicated that tumor associated antigens are inducing type 1 cytokines in similar fashion as LPS or colostrinin. However, lymphocytes taken from patients undergoing therapy with colostrinin revealed progressive loss capability to produce type 1 cytokines as they did in case of colostrinin. The loss of the capability to respond to antigen may represent phenomenon leading to immune tolerance.

Oral cavity gate of an entry into the communication network.

For ages naso-oro-pharyngeal cavity was considered as gate of entry to living organism for air and food. In recent years, however, the thoughts have changed considerably. Several lines of evidence indicate that the oral cavity with adjacent cavities plays a pivotal role for the recognition of signals coming from the surrounding world. These signals in form of food, germs, poisonous substances are initially analyzed in oral cavity and messages are distributed by a variety of communication pathways into pertinent parts of the body. All these diverse functions are performed by anatomical structures present in the naso-oro-pharyngeal cavity. The following article describes some of the functions discovered recently.

Oral application of cytokines.

A number of different laboratories reported on studies with orally administered interferons and cytokines. Their observations extend previous observations which showed that orally administered interferons and cytokines can exert both local and systemic effects. As difficult as it may be to understand how orally administered interferons and cytokines may exert both effects, the increasing number of laboratories that demonstrate biological effects with orally administered cytokines suggests that serious consideration be given to the possibility that orally administered interferons and cytokines can indeed exert effects. They also raise the possibility that these effects may have biological relevance for the treatment of human disease. Moreover, they may indicate that the nasal/oral region is a window on the environment. It is most important, however, to assure that these experiments are performed with special care to avoid presenting preliminary data that is not properly controlled. It is essential to carry out these studies with sufficient animals or patients to ascertain their significance; and to plan the studies as double-blind evaluations to avoid misinterpretations when subjective tests are used. Nevertheless, the overall data presented give one the impression of an area that should be pursued.

Natural human interferon alpha may act differently when given parenterally or orally to patients chronically infected with hepatitis B virus.

A literature review of patients chronically infected with hepatitis B virus (HBV) treated with natural human interferon alpha (nHuIFN-alpha) given parenterally every day for 28 days revelated that even the daily dose of (5 x 10(6)IU) of IFN-alpha inhibits cellular metabolism. As a result of metabolic block, the number of blood elements were diminished. Furthermore treated patients recorded several different adverse reactions. In contrast, among patients treated with the oral form of nHuIFN-alpha non metabolic block occurred and no adverse reactions were seen, even though the therapy lasted much longer. Two years after initiation of parenteral IFN-alpha therapy, the loss of HBV-BeAg was 53.8% and 28.8% of the patients had undergone seroconversion. In contrast, 77% of patients on oral interferon lost HBV-BeAg and 74% serconverted and normalized their biochemical liver function. The results suggest that the nHuIFN-alpha given orally and parenterally activate two different mechanisms responsible for virus elimination.

Natural human interferon alpha given orally has different effects on patients with distinct forms of chronic viral hepatitis B.

During therapy of chronic viral hepatitis B (CVHB), some patients treated with natural human interferon alpha (nHuIFN-alpha) lozenges failed to respond. These observations triggered studies aimed to determine whether there are markers predicting patients' response to therapy with nHuIFN-alpha lozenges. In these studies, 32 patients with CVHB were involved: 20 males and 12 females, 16-61 years of age with proven persistent hepatitis B viremia (HBV). Patients were evaluated for clinical, biochemical liver function, and virological markers of disease. During 300 days of treatment of the patients received 75-150 IU nHuIFN-alpha daily in form of lozenges. The responders to oral interferon therapy were those who had initially alanine amino transferase (ALAT) level higher than 100 IU (85.7% cure rate) and weak responses were observed among patients who had an initial ALAT level below 100 IU (9.0% response rate). Therefore, ALAT test in patients with CVHB may serve as a predicting indicator of the outcome of IFN lozenges therapy.

Comparison of the long-term effects of treatment with oral and parenteral interferon alpha in chronic viral hepatitis patients.

This report presents the interferon alpha (IFN-alpha) treatment results for 75 patients with chronic hepatitis B virus (HBV) (51 cases) and hepatitis C virus (HCV) (24 cases) induced hepatitis in maximal 61 months follow-up. Among the group of 51 patients with chronic HBV hepatitis, 35 were treated orally with IFN-alpha in the form of lozenges in low daily doses (37.5-150 U). The treatment was completed in 32 cases. The remaining 16 patients with chronic HBV hepatitis completed the treatment with parenteral IFN-alpha (3 x 10(6) U, 3 times a week). Positive results measured by the use of seroconversion in the HBe-antigen system were obtained for 68.7% (5-61 months follow-up) and 56.2% (7-44 months follow-up) of the patients treated with oral and parenteral IFN-alpha, respectively. Among the group of 24 patients with chronic HCV hepatitis, the first 6 patients were initially treated with IFN-alpha in the form of lozenges, in low daily doses. Biochemical remission was not achieved in these patients; genotype 1b was documented in 4 of them. Both, the first 6 patients (after a break) and the remaining 18 were treated with IFN-alpha parenterally, as in HBV patients. Temporary clinical and biochemical remission was achieved in 62.5% of the cases during the treatment, however the durable remission observed during 6-29 months of follow-up was achieved in 20.4 of the cases only.

Molecular species occurring in naturally induced interferon alpha.

Human interferon alpha (HuIFN-alpha) preparations induced in human peripheral white blood cells by Sendai virus were analyzed for activity after ultrafiltration, ion exchange, metal ion chromatography and isoelectrofocusing. It was found that HuIFN-alpha forms complexes with various physical properties and activities. Some of the IFN-alpha copurify with high molecular weight complexes. However, the low molecular weight fraction represent bulk of IFN activity. In isoelectrofocusing experiments, the former corresponds to IFN-alpha which possesses high pI (pH regions 8.5-10.5). An additional form of IFN-alpha are not complexed and possess a pI of 5.5-7.0. Our nIFN-alpha preparation preserve many of natural IFN isospecies and may have higher therapeutic value than the recombinant IFN-alpha preparations.

Oral therapy with human interferon alpha in calves experimentally injected with infectious bovine rhinotracheitis virus.

Fifty-six calves, seronegative for infectious bovine rhinotracheitis (IBR) virus, were randomly divided into 7 equal groups (n = 8) and given 0.0, 0.05, 0.50, or 5.00 international units (IU) of natural or recombinant human interferon alpha per kg body weight (nHuIFN-alpha or rHuIFN-alpha, respectively) orally once daily for 4 consecutive days, starting 2 days before intranasal inoculation with virulent IBR virus. Calves given 0.05 IU nHuIFN-alpha/kg bwt had significantly greater weight gain at days 15 (P < 0.10) and 25 (P < 0.05) than the placebo-treated (0.0 IU) control group. The treatment groups given 0.05 and 0.5 IU nHuIFN-alpha/kg bwt nHuIFN-alpha had fewer days with temperature > 40 degrees C (P < 0.05 and P = 0.10, respectively), and lower mean rectal temperatures on days 8 and 11 (0.05 IU/kg bwt; P < 0.10) or on day 11 (0.5 IU/kg bwt; P < 0.10). None of the calves given 0.05 IU nHuIFN-alpha/kg bwt required antibiotic therapy. Calves given 0.50 IU/kg bwt of nHuIFN-alpha, or 0.05 IU/kg bwt of rHuIFN-alpha had fewer (P < 0.05) total days of antibiotic therapy compared to controls. These data indicate that low dose oral IFN-alpha treatment significantly reduced the clinical effects of IBR virus infection in feedlot cattle in an interferon dose-dependent fashion.

Effect of low dose natural human interferon alpha given into the oral cavity on the recovery time and death loss in feedlot hospital pen cattle: a field study.

A total of 4577 feeder cattle were treated with a single oral dose of 33.0 international units (IU) of natural human interferon alpha (nHuIFN-alpha) per 100 pounds body weight, upon entry into the feedlot hospital pen. Another 2494 cattle received diluent alone and served as placebo controls. Cattle were evaluated on the number of treatment days required until the animals could be returned to the feedlot, and the death loss of cattle in the hospital pens. The mean number of days of treatment for control animals (3.9 days) was greater than in cattle treated with nHuIFN-alpha (3.1 days). Mortality of placebo-treated animals was 5.9% versus 3.6% (P < 0.001). These data suggest that oral low dose nHuIFN-alpha is of benefit in reduction of feedlot-associated morbidity and mortality.

The use of interferon modulates the negative effects of heat stress on poultry production.

A trial was conducted with broilers reared in two temperature environments; one was thermoneutral and the other had cycling ambient temperatures. Human interferon alpha (HuIFN-alpha) was added to the drinking water daily at four dose levels (0.0, 0.01, 0.1 and 1.0 international units (IU) per ml of drinking water). The trial began with 21 day old chicks housed either in a thermoneutral (24 degrees C) or a cycling ambient temperature (24-36 degrees C) environment. Interferon added to water at the highest concentration (1.0 IU/ml) improved surviability of birds in the cycling ambient temperature (24-36 degrees C) environment (P < 0.05). Birds housed in the cycling (24-36 degrees C) environment, drinking the lowest concentration of IFN-alpha 0.01 IU/ml, had a significantly improved weight gain-to-feed ratio. Oral IFN-alpha reduced the cost of production for birds reared in a cycling ambient temperature environment.

Evaluation of the efficacy of natural human interferon alpha lozenges on the clinical course of childhood neoplasia and in chronic hepatitis B virus infection in patients who were successfully treated for pediatric malignancies.

The immunostimulating and anti-cancer action of interferons (IFNs) has been known for many years. However, IFNs have not been introduced widely into the schemes of oncological treatment because of serious side effects potentiating untoward effects of chemotherapy. In addition using high doses of IFNs by parental routes the cost of such therapy is prohibitively high. Natural human interferon alpha lozenges produced from lymphoblastoid cell line by the Hayashibara Biochemical Lab. Okayama Japan (nHuIFN-alpha, HBL) is used in small doses delivered on oral mucosa. Thus, it might be expected not to cause severe side effects, and is less expensive. Children given antineoplastic and immunostimulatory treatment for cancer were also given nHuIFN-alpha--HBL lozenges containing 50-200 units of IFN per lozenge. Children treated age varied from 3-14 years. The average time of observation was 188 days. In 6 patients nHuIFN-alpha therapy was introduced at the time of the intensive oncological treatment during break periods. Those children had advanced malignant solid tumors. For the other children the IFN therapy was used after the successfully completed oncological treatment. The reason of using nHuIFN-alpha in this group was a long lasting hepatitis B virus antigenemia. The drug was well tolerated by children from both groups and a positive immunostimulating effect was observed. One prominent effect of the nHuIFN-alpha--HBL in children was a reduction of frequency of infections, improvement of appetite and psychological feeling of well being. It seems to us that IFN oral therapy may improve the tolerance of chemotherapy and radiotherapy.

Treatment of chronic viral hepatitis type B with oral mucosal administration of natural human interferon alpha lozenges.

Results of the administration of natural human interferon alpha (nIFN-alpha) into the oral cavity of 28 patients with chronic aggressive viral hepatitis type B are shown. Diagnosis of chronic aggressive viral hepatitis type B was based on clinical symptoms of disease, histopathological changes as evidenced by liver biopsy and persistence of HBV markers in patient sera. The daily dose of nIFN-alpha ranged from 75-200 IU/day. The twenty eight patients have been treated for a variable amount of time: thirteen over 300 days, two over 180 days, two over 120 days and eleven for less than 120 days. Only those patients who have been treated for over 300 days are considered to have completed the therapeutical program and remain under observation only. Oral IFN-alpha therapy is safe and efficacious in patients with chronic aggressive viral type B hepatitis. Among these 28 patients, 23 were initially positive for both hepatitis Bs antigen (HBsAg) and hepatitis Be antigen (HBeAg). Eight of these 23 patients have lost HBeAg and developed anti-HBe antibody. In addition one patient from this group seroconverted 356 days after initiation of treatment with IFN-alpha. Three patients lost HBs and HBe antigens and developed antibodies to both HBs and HBe antigens. Two patients who had eliminated HBe antigen before IFN-alpha therapy eliminated HBeAg following treatment and developed antibodies against HBs antigen. Three additional patients initially HBsAg+.HBcAg-, and HBeAg- developed antibody to HBe antigen during IFN-alpha therapy. At the time of this report 12 of the 23 initially viremic patients have seroconverted (52%).(ABSTRACT TRUNCATED AT 250 WORDS)

Treatment of six patients with chronic active HCV hepatitis, with low dose natural human interferon alpha administered orally.

Following the widely accepted therapeutic standard of treatment of HCV infection with parenteral interferon alpha, and encouraged by the author's won good experience with orally administered natural human interferon alpha in low doses (leuHuIFN alpha (ldou)), applied to chronic active HBV hepatitis patients, this form of interferon was given to six randomly selected HCV infected patients (2 women, 4 men) aged 34-62 years. The diagnosis was made based on a clinical and histological evaluation and confirmed by anti-HCV antibodies detection. In 2 out of 6 patients, leuHuIFN alpha (ldou) was employed immediately after steroid discontinuation. Patients were instructed to take one lozenge daily, in the morning, on an empty stomach, and keep it in the mouth until fully dissolved. Observation period varies from 19 to 69 weeks. In 3 patients the therapy concluded, after 19, 61 and 62 weeks, respectively. One patient after 4 weeks of treatment reported increasingly troublesome small joints pain and swelling, which forced leuHuIFN alpha (ldou) discontinuation after 19 weeks. In no patient transaminases normalization was seen during treatment; biochemical and clinical remission after the drug discontinuation was observed in only one patient, in whom the treatment was interrupted due to articular adverse symptoms. With HCV RNA levels assessment being unavailable at the moment, the treatment impact on the virus replication remains difficult to evaluate objectively. The treatment was well tolerated. All patients stressed significant increase of drive and appetite as well as improvement of the exercise tolerance.

Treatment of fourteen chronic active HBsAg+, HBeAg+ hepatitis patients with low dose natural human interferon alpha administered orally.

The therapy concept is based on a theory of the immunocorrective effect of orally administered natural human interferon alpha in low doses (leuHuIFN alpha (ldou)), manifolded by the logistic amplification system seated in the oral cavity. Fourteen randomly selected patients with chronic active type B hepatitis, aged 7-59 years, were assigned to treatment. All the patients had been treated for several months to several years with steroids, with no beneficial effect--clinical and biochemical symptoms of active liver disease, with histopathological progression (up to liver cirrhosis) had been permanently present. Treatment with leuHuIFN alpha (ldou) (doses: 50-100 U daily) was introduced immediately after the immunosuppression (steroids, steroids+azathioprine) discontinuation, and its influence on the course of the disease was monitored by means of hematological and biochemical tests, humoral and cellular immune response parameters, serological markers of HBV infection, HBV DNA concentration and immunohistochemical evaluation of liver biopsy specimens. The observation period ranges from 15 to 32 months. In all patients, within the first 3-6 weeks of treatment, transient deterioration of biochemical liver function tests was noted (e.g. 2-3 fold increase of ALAT), with no clinical symptoms of the disease exacerbation. The phenomenon lasted for 4-16 weeks. In all the treated patients, intensive immune system activation was seen, which lasted beyond the therapy period. Seven patients eliminated serum HBV DNA; all of them also eliminated HBeAg and seroconverted to HBeAb. Up to date, one person have lost serum HBsAg, in nine others its titre decreased significantly.

Early changes in the plasma proteins of patients treated with low doses of oral natural human interferon alpha (IFN-alpha).

The comparison of electrophoretic protein patterns and concentrations of the G, A and M immunoglobulins before initiation of oral IFN-alpha therapy of viral hepatitis and after 2 weeks of treatment allow to predict patient's response. Patient who after 2 weeks of treatment with ultra-low orally administered IFN-alpha responds with decrease of alpha-2 and beta globulin fractions, IgG levels and IgG:IgA ratio, together with increase of IgA and IgM concentrations has a good chance for rapid elimination of HBe antigen and seroconversion. On the other hand, a patient who responds to such therapy with decrease of serum albumin concentration, increase of alpha-1 and gamma globulin fractions in electrophoresis and decrease of IgG, IgA and IgM will most probably seroconvert rather late. Furthermore, a patient who responds with increase of IgG as well as IgG:IgA ratio and decrease of IgM and IgA:IgM ratio may be refractive to IFN-alpha therapy. Presented markers appear an important help for planning the therapeutic strategy for the chronically infected, HBsAg and HBeAg positive patients.

A new approach to chronic viral disease therapy by cytokines.

Eradication of an infectious agent during the acute phase of infection is a complicated process which involves several specific and non-specific mechanisms of the immune system. In chronic diseases, one of these steps may be missing or impaired which results in a malfunctions of the whole system and prolonged presence in the host invading pathogen. Mentioned above mechanisms are governed by cytokine network. Released by activated cells--cytokines initiate cascade reactions leading to eradication of pathogen. For practical purposes a model was formulated which resembles an electronic computer. In this model cytokines represent signals or words which when combined together form more or less precise instruction how to repair damages done by invading pathogen. Cells receiving cytokine signals become activated and transform messages increasing sometimes intensity of signals or release new ones which may initiate additional reactions. To activate full reaction two independent signals are required. Full activation of the eradication mechanisms requires activation of different types of cells. To achieve this goal proper combination of signals generated by cytokines should be induced. Efficacy of cytokines therapy may depend upon several factors such as route of administration, composition of cytokines, dose and modality of delivery. In the biological computer model introduction of cytokines on the surface of epithelium of the oral cavity in minute quantities and in proper composition should give the best clinical therapeutic results.

Inhibition of human immunodeficiency virus type 1 replication by human interferons alpha, beta and gamma.

The effect of various preparations of human interferon (HuIFN) upon human immunodeficiency virus (HIV-1) replication in cell lines and primary cultures of peripheral blood lymphocytes (PBL) was investigated. Natural interferon alpha (nHuIFN-alpha) exhibited a much higher inhibitory effect upon HIV-1 replication than did recombinant HuIFN-alpha (rHuIFN-alpha).