Lncs segmentation of erythema from skin photographs. Transmission of tuberculosis from adults to children in a. Abortive treatment of syphilis, contribution. various forms, from eczema to anaphylactic reactions with possible lethal through cutaneous tests: prick-tests or IDR (intra dermo reaction). All the animals had negatiVe intra dermo reaction to bovine tuberculin ( IU in ml), and a negative serology to Brucella. All the bovines were also.
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A therapeutic vaccine peptide complex for preventing or treating disorders in mammals and particularly in humans, in Canidae, Felidae and Equidae whose protective immunity depends on the stimulation of type Th1 lymphocytes and in particular a delayed state of hypersensitivity. The vaccine peptide complex contains the following amino acid sequence A16E: Therapeutic peptide vaccine complex designed for the prevention and treatment of conditions in mammals and in particular, in humans, canines, felidae, and equidae whose protective immunity depends on the stimulation of T lymphocytes of the Th1 type and notably of a state of hypersensitivity of the retarded type.
During the immune response, the T lymphocyte comprises an important source of cytokines. Their production is induced after stimulation in a specific manner in the presence of antigens or in a non-specific manner in the presence of mitogens concanaviline A, phytohemagglutinin.
It is possible to divide, in a very simplified way, the immune responses into two large qualitatively distinct categories, humoral responses which bring into play the production of antibodies by the B lymphocytes, and cellular responses retarded hypersensitivity reaction, cytotoxic reactionfor which the effective cells are the T lymphocytes.
These auxiliary T cells recognize the antigen in combination with the class II molecules of the major histocompatibility complex. A major concept emerged in when T. Th 1 cells for T helpers of type 1 and Th 2 cells for T helpers of type 2. The example of leishmaniasis will be used, which is a parasitic infection endemic, or even epidemic, in tropical and subtropical regions of the world.
The leishmaniaflagellate protozoa of the family of trypsanosomatidae and of the genus Leishmaniaare the pathogenic agents responsible for these diseases.
Numerous studies concerning the immune responses during experimental murine leishmaniases have led to the demonstration of the predominant role of cell-mediated immunity and the existence of a duality of the immunological response.
There are fundamentally two types of responses against leishmanias: It has thus been demonstrated that the subpopulation of auxiliary T lymphocytes of the Th1 type producer of interferon gamma and interleukine 2 was capable of eliminating the amastigote intracellular forms by means of the activation of macrophages Reiner S. Conversely, the subpopulation of auxiliary T lymphocytes of the Th2 type producer of interleukine 4 is responsible for exacerbating the disease.
In humans, certain facts are comparable by nature. Only one study led by Pinelli et al. A good vaccine candidate must thus match one or more strongly immunogenic parasitic antigens capable either of blocking the differentiation of the Th2 lymphocytes Gurunathan S et al. Exp Med, Oct. Planning to vaccinate against leishmanias is still problematic today. It can be cited that the use of living parasites, irradiated parasites, and completely killed parasites Moreau Y et coll.
In the s, purified extracts of parasitic antigens were used in dogs in inducing an exacerbation of the disease: LIF2 fraction and anti-idiotypical vaccine from Dr. Currently, several molecules are in trials and a final result is pending. C et al, J of Immunol, However, none of the current immunization protocols make it possible to obtain a sufficient level of protection or in any case, it is not reproducible.
To date, no work has been done with synthetic peptides. Reactuon present invention comprises an immunomodulator complex that uses one or two reactuon with an adjuvant inducing either an immunostimulation of the lymphocytary system T of the type Th1 in a reproducible manner, or an immunomodulation of lymphocytes of the type Th2 into a type Th1. It is important to emphasize two essential facts.
Firstly, the Th1 and Th2 cells exert, by means of certain cytokines that they produce, a reciprocal counter-regulation effect, which explains why in the majority of these cases, an alternation of the cell-mediated and humoral responses is observed.
Thus, the immune responses that favor a reaction of the Derko lymphocytes of the Th1 type, i. Conversely, a response that favors the production of antibodies will be most often associated either with the participation of the T lymphocytes of the Th2 type or with a relative deficiency of the specific cellular immunity; this is the case of visceral leishmaniasis.
The second fundamental circumstance is that lymphocytes fully differentiated into cells of the Th1 and Th2 type are not pre-existing in the intea simple host. The polarization of the immune responses into a Th1 or Th2 phenotype has been ijtra with numerous pathological situations. For infections by leishmania, Typanozoma, Candidaand other intracellular organisms vermo as Mycobacterium and Listeriaa Th1 response correlates to resistance to the pathogen.
On the other hand, certain conditions such as canine atopic dermatitis, allergies and asthma lead to the exacerbation of a response of the Th2 type. The reactioon to immunostimulants which permit the passage from a Th2 response to a Th1 response, thus the passage from a state of immediate hypersensitivity to a state of hypersensitivity of the retarded type, must lead to a recovery.
It can be considered that the example of asthma, for which the medical community is worrying about the increasing number of individuals who suffer from it: In France, 2, people die from reavtion every year. Inyra is a chronic inflammatory condition of the respiratory tracts where numerous cells of the immune system are intervening.
The disease becomes chronic because of the quasi-permanent accumulation of inflammation mediators, as well as the quasi-incessant recruitment of destructive polynuclear reavtion The damage caused by the basic proteins leads to a hyperactivity of the bronchial tubes: It can be recalled that the activated T lymphocytes release notably interleukine 4, which starts the production of the immunoglobulins E.
The gamma interferon activates the cells which have the antigen, and which, in turn, produce interleukine Thus, interleukine 4 orients the immune reactions towards the allergic reactions, while gamma interferon seems to promote the reactions of deaction defensive type.
And yet this orientation of the immune system will be set very early, in the very young infant. In a newborn baby exposed to a pathogenic agent, the immune system becomes oriented to the defensive method: These children will have less allergic risks. On the other hand, newborn babies raised in a sterile setting have risks of being confronted with an environment high in allergens and low in pathogenic micro-organisms.
If this is the case, the still immature immune system will activate the T lymphocytes which secrete interleukine 4, giving the immune system an allergic orientation, which favors asthma. A reorientation of the immune reactions of sensitive persons, by limiting the allergic Th2 method of immune response, to an activation of the Th1 method would be a possibility for the treatment rection asthma. It can be extended that this phenomenon to other forms of immediate hypersensitivity.
Immediate hypersensitivity is the most frequent form of allergy and results from the synthesis of immunoglobulins E IgEinrra specific to the allergens from the environment. These immunoglobulins E become attached to the cells whose mastocytes are the key elements. The contact of the allergen drrmo the immunoglobulins E activates the mastocytes which release the mediators of the inflammation: Grouped under the name of anaphylaxia and atopic diseases are all clinical manifestations linked to the phenomena of immediate hypersensitivity which result from the production of immunoglobulins E, the great culprits rexction the allergy.
However, anaphylaxia corresponds to a physiopathological mechanism independent of any hereditary factor it is, for example, the anaphylactic shock due to hymenopteran venomswhile atopia is the expression of a particular genetic aptitude to produce immunoglobulins E in excess directed against various natural substances of the atmospheric environment pollens, molds, pollutantshousehold environment dust mites, cockroaches, mammals or professional environment, and also against foods; this results in respiratory allergies asthma, imtracutaneous allergies urticaria, atopic eczemaophthalmic allergies and digestive allergies.
In these pathologies, two points must be mentioned:. Today, treatments that confine the syndromes having an allergic origin comprise a part of the anti-inflammatory arsenal anti-histamine and corticoids.
The predominant place taken by the cytokines in the fundamental mechanisms teaction immediate hypersensitivity opens prospects for new targeted therapies: The invention also involves the use of these fragments and the adjuvant as diagnostic reagents in vitro and in vivo, and as reagents that induce a stimulation of the T lymphocytes of the Th1 type ensuring an ibtra of the development of the lymphocytes of the Th2 type, notably for the prevention and the treatment of conditions linked to a condition of immediate hypersensitivity, i.
The two peptides A16E and A16G have the following sequences of amino acids: The peptide compound according to the invention can also be comprised of possible derivatives of the peptides A16E and A16G, reqction derivatives comprising at least five adjoining amino acids taken in the sequences A16E or A16G.
The other possible derivatives are: The peptide compound according to the invention can also be included in any polypeptide. The peptides obtained by synthesis and their derivatives are constructed in octopus or are made immunogenic by connections to the carriers large molecules of the KLH typeand are administered to mammals in the presence of an adjuvant, preferably muramyl dipeptide MDP: The mechanism of specific action of the peptide complex obtained according to the invention is verified using traditional methods that allow the dosage of peptides, their identification, and using more specific methods that show that the innovative peptide complex takes effect either by immunostimulation of the lymphocyte system of the Th1 type, or by immunomodulation of a Th2 type towards a Th1 type.
A parasitological exam was done from the inntra taken directly from the candidate studied, a dog, for example. A smear of a puncture of the bone marrow is made on a slide. Samples of the bone marrow are brought under cultivation in reactiob biphase cultivation medium NNN Novy and Mac Neal,4.
The blind subculturings were done every four to six days.
The parasitemy was quantified as follows: Evidence of the involvement of a cell-mediated immunity of the Th1 type:. Leishmanias of the promastigote forms are cultivated in the standard culture media. Parasites are harvested from the end of the exponential phase days. This constitutes the leishmanias for the intradermoreaction test IDR. The study of the immune response of the Th1 type that follows was performed on dogs.
Dogs are placed in lateral decubitus and a delicate and non-irritating shearing is done on the thoracic zone approximately 5 cm by 10 cm behind the elbow. Four circles 10 mm in diameter are marked using a felt-tip pen.
Tests cutanés tuberculiniques
Into the center of the circles, 0. Two circles receive the solution of leishmanias and the 2 other circles receive the saline methiolated solution in the negative control. The test is considered positive if the mean of the two observed induration diameters is greater than or equal to 5 mm.
The observation of an erythema without induration will be considered to be a negative test Pinelli et al.
The present invention thus also involves a product for in vivo diagnosis which reveals a retarded hypersensitivity immune condition of the Th1 type using peptides A16E and A16G and their derivatives in an intra-dermo reaction in mammals. One can also carry out dosage of nitrogen monoxide NO defmo know the destructive activity of the monocytes against the Leishmanias. NO has a high chemical reactivity.
Tests cutanés tuberculiniques
In the presence of water and oxygen, this molecule is rapidly oxidized in a stochiometric manner and thus forms the nitrites NO2- according to the reaction: The reacttion accumulate in the media and are easily detectable chemically by the Griess method.
The colorimetric reaction develops in the dark for 2 minutes.
The optical densities obtained at nm are corrected by the subtraction of the OD obtained on the wells containing only the culture medium.
For this test, the monocytes and lymphocytes are isolated from the venous blood of dogs. After 3 days of cultivation, the macrophages are washed in RPMI complete medium, supplemented dermi fresh medium.
When they are used, the lymphocytes cultivated separately are washed, counted, and added to the macrophages in the ratio of 2 lymphocytes per macrophage. Next, a test is done on the lymphocyte proliferation. These cells are brought under cultivation on a plate having 96 wells at a concentration of 2. The optimal antigen and eermo concentrations have been determined in prior experiments. All of the tests are done in triplicate. In our experiments, the BrdU is added for 18 hours after 72 hours of incubation.
The cells which have incorporated the BrdU in their ADN are easily detectable in the presence of a monoclonal antibody directed against the BrdU.