IMMUNOSUPPRESSION – BLISS AND CURSE: a tale of two poisons

NK Mathur and Puneet Bhargava

Department of Dermatology, Leprosy and STD

SMS Medical College and Hospital

Jaipur

In the present era, monitored immunosuppression by Cyclosporin-A(CsA) is a bliss for organ transplant patients, while much similar, but unabated, immunosuppression by Human Immunodeficiency Virus (HIV) is no less than a curse on mankind. This review is a collection of similarities in the actions of these agents on human systems. As a back drop of these common toxicities, we have developed a drug based on principles of Homoeopathy.

HISTORICAL BACKGROUND

Cyclosporin A (Cs-A) is a neutral, homodetic, hydrophobic, cyclic 11- peptide isolated from Tolypocladium inflatum Gams fungal species.1 It is widely used following organ transplant due to its unique and remarkably effective immunosuppressive properties. The credit of its discovery goes to Sandoz laboratory, Switzerland, where in early 1970, they took up the study of metabolites from various soil fungi to study their anti-bacterial and anti-mitotic properties.1 Tolypocladium inflatum Gams was one of them. Ruegger extracted a mixture of cyclosporins and named it compound 24-556.1 In 1971, Borel and his team started working on the immunosuppressive properties of 24-556 and by early 1973 demonstrated that 24-556 suppresses both antibody production and cell mediated immunity. Later, Reugger separated CsA from compound 24-556.2 Soon, Borel et al (1976) described the antilymphocytic properties of CsA. Another milestone was the year 1978 when first clinical trials of CsA were conducted in patients of kidney transplantation.1 While CsA was under development, HIV, the causative agent of AIDS was brewing some where in central Africa. The oldest evidence of its infection comes from a study by Saxinger et al, where they examined sera obtained from a 1972 study of Ugandan children and found that 65% of apparently healthy children had sera positive for HIV.3 The year 1981 signified the arrival of HIV infection in USA, when a number of reports appeared describing unexpected deaths among young homosexual men caused by Kaposi’s sarcoma, unexplained opportunistic infections, or both.4,5 It was not until 1983 that HIV was actually isolated.6,7 Similarly 1983 was an important year for CsA also as it was in this year that CsA was first approved and registered for transplant patients1 in USA, Switzerland and some other major countries. The first enzyme immunoassay to detect antibodies to HIV was licensed in 1985, 8 while the monoclonal antibodies against CsA were developed in 1987. 9

STRUCTURE

CsA and HIV-1 gag share structural similarities, as both CsA and gag capsid protein structure consists of eight stranded b – barrels which have binding sites for intracellular proteins; cyclophilins, the peptidyl-prolyl-cis-trans isomerases, which assist in the intracellular folding of proteins.10 CsA-cyclophilin-A complex binds to calcineurin contributing to immunosuppression produced by CsA. Gag-cyclophilin combination helps in viral replication and although this complex does not bind to calcineurin, it may contribute to HIV induced immunosuppression by inhibiting the binding of the cyclophilins to a possible physiological ligand.10

CELLS OF IMMUNE SYSTEM

T-Cell : CD4+ T lymphocytes are the key regulators in the immunosuppression produced by HIV and Cyclosporin.11-14 Both HIV and CsA interfere with intracellular pathways in T-cell activation15-20 and depletion of these cells.

Intracellular signaling pathways in T-cell activation (fig1):

T-cell activation process begins with the association of peptide-MHC complex with TCR-CD4 molecule. This association activates intracellular phosphotyrosine kinases (PTKs). Three different PTKs have been implicated in early phosphorylation events (lck associated with cytoplasmic tail of CD4 and fyn and zap kinase with z -chain of TCR complex). These PTKs phosphorylate tyrosine moieties of the enzyme phosphatidylinositol phospholipase C-g 1(PI-PLC-g 1) and activate it. PI-PLC-g 1 catalyses hydrolysis of plasma membrane phospholipid called phosphatidylinositol 4,5 biphosphate to Inositol 1,4,5 triphosphate (IP3) and diacylglycerol (DAG). IP3 stimulates release of membrane sequestered intracellular calcium, which then forms complex with a calcium dependent regulatory protein called calmodulin. Calcium – calmodulin complex activates a phosphatase Calcineurin. Calcineurin dephosphorylates cytoplasmic component of NFAT (nuclear factor of activated T-Cells) called NFATc and permits it to enter the nucleus. Diacylglycerol activates protein kinase C (a serine/threonine protein phospho kinase). This activation leads tofos/jun synthesis and AP-1 (another nuclear factor) generation. Inside the nucleus NFATC, combines with newly synthesised AP-1 proteins and this complex binds to NFAT binding sequences of the IL-2 promoter, leading to increased IL-2 transcription and cellular activation.21-23 PKC also phosphorylates Ik B, an inhibitor of NFk B (Nuclear factor of immunoglobulin k light chains in B cells) to activate NFk B, which is then translocated to the nucleus and binds to IL-2 promoter site to increase IL-2 transcription.21-23 IL-2 promoter contains additional binding site for another factor NFIL-2A, composed of 2 proteins OCT1 and OAP. NFIL-2A activation occurs through TCR but the final pathway is not clear.21-23Cyclosporin inhibits this cycle at multiple steps. It binds to a family of intracellular proteins called cyclophilins (CyPs).15 Cyclophilins have the ability to catalyse cis-trans intra conversion of peptidyl-prolyl bonds and promote protein folding in cells.24,25 Cyclosporin-cyclophilin combination binds to calcineurin and inhibits its activity.16,17 In addition CsA binds to calmodulin and inhibits its ability to activate calcineurin.18Further CsA also interferes with the translocation of NFATC in the nucleus 26,27 and its binding to the IL-2 promoter.28 Translocation of NFk B to the nucleus and its binding to IL-2 promoter is also CsA sensitive.29,30 At the same time, induction of NFIL-2A DNA binding activity is also inhibitable by CsA.17 Although the knowledge about how HIV affects this cycle is incomplete, it appears that binding of gp 120 to CD4 disrupts the earliest events in signal transduction. This disruption may result from activation of lck through CD4 before TCR signaling, which may result in a negative signal, 31 sequestration of lck, 32 activation of a negative regulatory kinase such as csk or inactivation of CD45, a key regulator of early T cell activation events.19 Binding of HIV gag to cyclophilins may inhibit cellular activation and induce immunosuppression in a manner similar to CsA.33Gupta S (1993) has hypothesised after studying HIV infected mononuclear cells, that the defect may lie downstream of PKC.20Glutathione deficiency may also contribute to defective T-cell signal transduction by altering cellular redox potentials in HIV-AIDS.19

CD4+ cell functions: Dysregulation of CD4+ helper cell functions occurs long before depletion of these cells34-35 in HIV-AIDS . Acute primary phase in HIV-1 infection is characterised by low serum IL-2 concentration.36 With progression and HIV seroconversion, a sequential loss of TH functions is seen; earliest being response to recall antigens such as influenza A virus, tetanus toxoid and HIV synthetic peptides, followed by the allo- major histocompatibility complex and finally by phytohemagglutinin (PHA).37 Similarly, IL-2 and lymphokine production are the most sensitive of all lymphocyte functions to CsA (50% inhibitory concentration 10-20 ng/ml), followed by cellular proliferation (20-50 ng/ml), response to CON A (200-500 ng/ml) and PHA (500-1000 ng/ml).38-39

CD4+ cell count and mechanisms of depletion: HIV-AIDS shows a rapid decline in CD4 cell counts with disease progression.40-41 Although cyclosporin at the normal maintenance dose (100-200 ng/ml) is not lymphotoxic,38-39 it may share a number of mechanisms of cell death with HIV at higher concentration. Apoptosis is one of the main mechanisms of cell death in HIV infection. HIV tat upregulates CD95 ligand expession in CD4+ cells and sensitizes them to TCR and CD4 induced apoptosis by soluble gp 120.42 Similarly, Huss et al (1995) observed that CsA induces apoptosis in canine CD4+ lymphocytes in a time and dose dependent fashion over a range of 6 to 10 m g/ 100 m l.43 Encoded retroviral products and microbes such as S. aureus can act as superantigens (a group of microbial antigens which can bind to specific Vb determinants on T cells resulting in massive stimulation, followed by deletion or anergy of these cells) and contribute to HIV induced immunosuppression.44-47Similarly CsA can promote superantigen mediated cell death in CD4+ lymphocytes.48 Experimental and clinical evidence suggests that CD8+ suppressor cells play a role in AIDS progression by causing CD4+ helper cell depletion.49 Likewise, generation of suppressor cells and reversal of TH:TS ratio is seen after CsA therapy.50 Other mechanisms of depletion of CD4+ lymphocytes in HIV infection are direct cytopathic effect by HIV, formation of multinucleated giant cells or syncytia, uninfected CD4+ cell destruction by antibody dependent cellular cytotoxicity, NK cells and HIV-specific cytolytic T lymphocytes and autoimmune mechanisms.51-52

Macrophage/monocyte: Cells of monocyte /macrophage lineage are among the primary cells infected by HIV53and form its major reservoir of HIV.54-55 Although,mild functional abnormalities such as defects in chemotaxis, secretion of IL-l, FC receptor functions, oxidative burst responses, C3 receptor mediated clearance, certain cytotoxic functions and most importantly, defective antigen presentation and induction of T-cell responses are seen in HIV-AIDS,56-57 most studies have shown that changes in blood monocyte number, phenotype and functions are minimal even in late stage disease.55,58 Cyclosporin-A directly interferes with antigen presenting function of macrophages59-61,while other functions such as phagocytic (in vitro or in vivo) or migratory activity and LPS induced IL-1 production are insensitive to drug concentrations that markedly affects T cell functions.62

Dendritic cells(DC) including Langerhans cells: HIV infects Langerhans cells and peripheral blood dendritic cells both in vitro and in vivo. 63-66 Low dose viral exposure generates protective cytotoxic cell response directed against a variety of epitopes of HIV-1.67 At higher viral load and longer time of exposure, antigen presenting functions of DC are compromised;68 reduced classII and ATPase levels being the first indicator of altered Langerhans cell functions;69 and DCs lose their capacity to stimulate antiviral responses in T cells.70 Very high viral load in mature DCs results in syncytium formation and death of DCs and of any clustered T cells.71 Similarly in vitro studies have shown that CsA inhibits the accessory and antigen- presenting functions of Langerhans cells, directly in a dose dependent manner.72

B-cells: Cyclosporin can inhibit directly the activation of B-lymphocytes to a variety of stimuli by interfering with Ca++ dependent signals.62Similarly functional B-cell defects are also seen in HIV infection.73 Lun et al (1991) have shown that along with inhibiting B cell activation, CsA may also decrease or increase the differentiation and Ig production by B cells, depending on the antigen used.74 Similarly HIV is also a B-cell mitogen and induces polyclonal B-cell activation and Ig production by the cells.75

Natural Killer cells: Accumulated evidence indicates that NK cell responses are normal or elevated in early phases of AIDS,76-77 however progressive decline in function are noted with increasing duration of HIV infection.78 Mechanisms may include reduction in circulating levels of IL-2 required for proliferation of NK cells, or inhibition of cellular functions by HIV-1 viral peptides.79 NK cell activity is only mildly impaired under normal CsA therapeutic dose.80 However, a dose dependent inhibition of NK cell activity is noted when NK cells are exposed in vitro to CsA.81

Mast cells: Animal studies have shown that CsA directly leads to depletion of mast cells and reduces secretion of mast cell specific proteases.82 Triggiani et al (1989) showed that CsA inhibits release of histamine and prostaglandin D2 from human lung mast cells.83 Similarly, decreased skin reactivity to codeine in patients with AIDS is due to decrease of local mast cell density or releasibility.84

METABOLIC ABNORMALITIES

Glutathione deficiency: Glutathione is an important antioxidant which protects the body from free radical injury. HIV infection produces systemic glutathione deficiency 85 which correlates strongly with low CD4 count, impaired IL-2 production and proliferation of peripheral blood mononuclear cells, 86 and increased incidence of adverse reactions to Co-trimoxazole due to failure to detoxify its hydroxylamine derivatives.87CsA also produces glutathione deficiency and the resultant oxidative stress contributes to its hepatotoxicity and nephrotoxicity.88-89

TRACE ELEMENTS:

Selenium: Selenium is an important component of glutathione peroxidase, which is depleted in HIV positive and CsA treated individuals.90-92Selenium levels are low in patients with AIDS and this deficiency may be associated with myopathy, cardiomyopathy, immune dysfunctions, decreased T-cell counts and impaired phagocytic functions91seen in these patients. In CsA treated patients, selenium supplementation seems to prevent the vascular toxicity.92

Magnesium: Cyclosporin therapy has been associated with renal magnesium wasting and hypomagnesemia. 93 Belief is that this drug induced hypomagnesemia may lead to hypertension,94 contribute to neurotoxicity95 and may exacerbate cyclosporin induced nephrotoxicity.96-97Similarly low magnesium levels are found in HIV-AIDS patients.98-101 There is direct co-relation between hypomagnesemia and absolute CD4counts.98,101 Further, low magnesium level may lead to lethargy, impaired mentation99 and convulsive status epilepticus100 in these patients.

Zinc: Low plasma Zinc levels are found in both HIV infected102 and CsA treated individuals,103 although high levels have also been reported after CsA therapy.104

Lipid Metabolism: Immunosuppressive therapy with CsA has been associated with significant increase in LDL cholesterol.105 On long term immunosuppresion, this hyper-cholesterolemia may be a potential risk for atherosclerosis. Similarly HIV-AIDS patients have been shown to have a large number of lipid metabolism abnormalities including abnormally elevated triglyceride levels,106 raised fatty acid turnover,107 raised fat oxidation rates unrelated to counter-regulatory or thyroid hormone increase108 and relative loss of lean body mass compared with fat.109

ORGAN SYSTEM ABNORMALITIES

Thymus: Thymus is an important organ for T cell maturation and differentiation. Thymocytes develop into T cells in the thymic microenvironment consisting of epithelial cells, macrophages and interdigitating cells (IDCs) which produce different cytokines involved in thymocyte maturation.110-111 Both HIV and CsA are known to affect thymus. 112-113 HIV infection produces rapid involution of thymus.114Similarly CsA causes a marked reduction in the size, weight and consistency of the organ.115 Microscopy shows that in HIV infection, there is fatty replacement of thymus with loss of corticomedullary junction, paucity of Hassall’s corpuscles and depletion of lymphocytes and keratin positive epithelial cells.114 Studies in mice have shown that CsA also results in loss of medulla, loss of keratin positive epithelial cells, Hassall’s corpuscles and lymphocytes.116 Study of the cell types affected shows that both HIV and CsA cause depletion and prevent maturation of immature CD4+ CD8+ (DP) cells into CD4+ CD8- and CD8+ CD4- (SP) cells.112,113,117 This effect occurs by similar mechanism like accleration of apoptosis by both HIV and CsA.112,116,117 Further CsA disrupts thymic micro- environment.116 Similarly HIV infection of thymic macrophages, dendritic cells and epithelial cells may compromise their functions in regulating thymopoiesis.118,119 This compromise on the functions of thymic microenvironment may further contribute to the depletion of CD4+ CD8+ (DP) cells 112,113 by both HIV and CsA.

Central Nervous System function: AIDS dementia complex or HIV encephalopathy is probably the most common CNS complication of HIV infection.120 Patients present with a decline in cognitive ability from a previous level, along with motor and behavioural dysfunctions.56Mechanisms include gp120 mediated neuronal toxicity, myelin damage produced by viruses other than HIV (measles, herpes simplex and JC virus), destruction of white matter by host immune responses, and possible involvement of quinolinic acid and TNF-a .121-125 Similarly cyclosporin therapy is associated with both reversible and irreversible dementia. This pathology arises secondarily to cyclosporin induced hypertension or vascular injury.126-130 Both partial and generalised seizures complicate HIV-AIDS, probably as a consequence of HIV encephalopathy, opportunistic infections and neoplasms.120,131,132 Similary, seizures are a common side effect of CsA therapy.133-136 Although multiple factors are involved, they probably arise as a result of cyclosporin induced lower seizure threshold.135 Peripheral neuropathies including distal sensory polyneuropathy, mononeuritis multiplex, isolated mononeuropathy, polyradiculopathy and acute inflammatory demyelinating neuropathy (Guillain-Barre syndrome) arise in HIV infection.137-140 Probable mechanisms are related to HIV mediated axonal degeneration, autoimmunity, associated infections (CMV and Mycobacterium avium intracellulare) and Vit B12 deficiency.137-142 Similarly, CsA therapy is associated with a demyelinating polyneuropathy.143-145 Cyclosporin also enhances virally induced T-cell-mediated demyelination.145There are also isolated reports of Guillain-Barre syndrome arising following immunosuppression involving cyclosporin.146-147 Wasserstein et al (1996) observed parkinsonism in two patients receiving cyclosporin.148 One patient improved with carbidopa-levodopa therapy and other with reduction of cyclosporin dosage.148 Similarly, parkinsonian symptoms with markedly reduced dopamine concentration in caudate nucleus are seen in HIV-AIDS.149-150

Neuro-Psychiatric Manifestations: Reversible visual hallucinations may occur in a dose dependent manner following cyclosporin therapy.151-152 Similarly, hallucinations may be a part of psychosis associated with HIV infection.153-154

Renal abnormalities: Both HIV infection and CsA produce renal abnormalities.155-159 About 10% of HIV-1 infected patients develop a chronic renal disease characterised by tubular changes (microcystic tubular dilation and regeneration), interstitial inflammatory infiltrate and more commonly, focal segmental and global glomerulosclerosis (FSGS). This entity is termed as HIV associated nephropathy.155-156 CsA, similarly produces renal side effects, but in a dose dependent manner,157 including tubular changes (isometric vacuoles, inclusion bodies, microcalcifications), striped interstitial fibrosis and more seriously, focal and segmental glomerulosclerosis.157-159 Recent evidence shows that Transforming growth factor-b (TGF-b ) may be involved in HIV associated FSGS.160 HIV-1 tat has been shown to increase the release of TGF-b 1 from bone marrow macrophages.161 Similarly, cyclosporin has been shown to enhance the expression of TGF-b , both in vivo and in vitro162and TGF-b has been implicated in CsA augmented fibrogenesis,158 although angiotensin II may also be involved in this process.163

Cardiac abnormalities: Although there has been controversy whether HIV directly affects heart, cardiac lesions have been found in both HIV infection and after cyclosporin therapy.164-169 CsA in a dose dependent manner produces perimyocytic fibrosis164 and severe myocardial calcification.165-166 Similarly, autopsy studies have shown that cardiomyopathic changes like myocyte necrosis, myocyte hypertrophy and diffuse myocardial fibrosis are common in AIDS.167-168 Calcification in the media of most major vessels is typical of HIV arteriopathy.169

Hypertension: Systemic hypertension is a frequent side effect of CsA therapy.170-172 Mechanisms include activation of endothelin system,173sympathetic nervous system174 and renin angiotensin system.175 Although systemic hypertension is not seen, plexiform variant of pulmonary arterial hypertension related to chronic HIV infection does occurs in HIV- AIDS.176

Testicular functions: Both CsA and HIV affect testicular functions.177-184 Cyclosporin induces a dose dependent decrease in serum and intratesticular testosterone levels and produces impairment of testicular spermatogenesis, steroidogenesis, epididymal sperm maturation and fertility in rats.177-178 These effects are reversible on discontinuing CsA therapy.179 Mechanisms include direct inhibition by CsA of testosterone biosynthesis at multiple sites, interference with signal transduction pathways180and suppression of hypothalamo-pituitary axis.177 Similarly, hypogonadism is seen in approximately 50% HIV infected individuals.56 Laudat et al (1995) showed that hypogonadism occurs as the CD4 lymphocytes decrease and dehydroepiandrosterone (DHEA), androstenedione, dihydrotestosterone (DHT)and non-SHBG testosterone levels correlate with CD4 counts.181 Probable factors leading to hypogonadism include peripheral inhibition of testosterone biosynthesis by HIV, opportunistic infections, chronic debilitating illnesses and suppression of hypothalamo- pituitary axis by cytokines.182-184

Hepatobiliary system : Cholestasis, manifested by increased levels in serum bilirubin, alkaline phosphatase and gamma-glutamyl transferase, is the most frequent manifestation of CsA hepatotoxicity.185-186 It is dose dependent and is often associated with a variable component of cholangitis and pericholangitis. It is probably mediated through non- competitive binding of CsA to the bile acid transporter at the cell membrane.187-189 Similarly, cholestasis as a component of biliary disease occurs in patients with CD4 counts less than 100 per mm3 in AIDS.190 The underlying causes may be related to papillary stenosis, cholecystitis, sclerosing cholangitis and intrahepatic abnormalities.191

Pancreatic functions: Cyclosporin, when administered to rats in therapeutic doses, leads to glucose intolerance, hyperglycemia, hypoinsulinemia and marked decrease in pancreatic insulin content.192-195 Accumulated evidence indicates that CsA has a direct toxic effect on islet b cell functions, leading to impaired insulin synthesis and secretion.196-199 Ultrastructurally, varying degrees of vacuolization and dilation of endoplasmic reticulum and golgi apparatus can be observed in b cells accompanied by reduction in the number of secretory granules.200-201Similarly, pancreatitis leading to abnormalities of glucose metabolism are seen in HIV infection.56,202-204 Probable mechanisms of pancreatic dysfunction include drug toxicity by pentamidine and dideoxynucleosides, opportunistic infection (CMV, MAC, candida, cryptosporidia ) and neoplasms.202-204

Gingival involvement: Gingival hyperplasia occurs frequently after CsA therapy.205-206 Similarly human immunodeficiency virus – associated gingivitis (HIV-G) is a frequent finding among oral manifestations of HIV infection.207-209

Muscular System: HIV associated myopathy, now a well defined condition, ranges from an asymptomatic elevation in creatine kinase to a subacute syndrome characterised by proximal muscle weakness and myalgias.56,210-212 It may occur at any point of HIV-1 disease.210Cyclosporin therapy, too, is associated with a dose dependent toxic myopathy characterised by myalgias, muscle weakness or rhabdomyolysis.213-215

Pulmonary function: Recent reports have documented the occurrence of adult respiratory distress syndrome (ARDS) after cyclosporin therapy.216-217 It is believed that high concentration of cyclosporin in the pulmonary vasculature causes a localized ‘capillary leak’216 or it may be an idiosyncratic reaction to cyclosporin.217 ARDS also occurs in HIV-AIDS, probably as a manifestation of diffuse lung injury produced by toxoplasmosis,218 pneumocystis carinii pneumonia,219 cryptococcosis,220 pulmonary tuberculosis221 and varicella infection.222

Hemolytic Uremic Syndrome and Thrombotic thrombocytopenic purpura: Cyclosporin therapy is related to the development of a syndrome resembling thrombotic thrombocytopenic purpura (TTP) or hemolytic uremic syndrome (HUS).223-227 Probable mechanisms include defective synthesis and release of prostacyclin from endothelial cells,228 and excessive platelet aggregation due to increased utilization of high molecular weight multimers of factor VIII, whose levels are raised after vascular injury induced by cyclosporin.229 Complications, HUS and TTP, are also seen in patients of HIV infection. These lesions probably arise secondary to HIV infection induced endothelial injury.230-235

Hypertrichosis: Hypertrichosis of eyelashes has been recently described as a cutaneous marker for AIDS .236 Like wise, trichomegaly237-239and generalised hypertrichosis is one of the common side effects of CsA , seen not only in normal, but also in pathological conditions of hair growth such as alopecia areata and in some patients of male pattern alopecia.240 Hair growth by both CsA and HIV is probably due to direct stimulant effect on hair follicle keratinocytes 241-243 or secondary to immune dysregulation.242

INFECTION: Basic defects in both HIV infection and CsA treated patients include T cell functions, macrophage and NK cell activity. Therefore, it is expected that intracellular pathogens should be common in both HIV infected and CsA treated individuals. Both the primary and reactivating CMV diseases are common in CsA treated patients.244-245 CMV retinitis occurs in HIV infected individuals and is an AIDS defining illness.56 EBV induced lymphoproliferative lesions occur both in AIDS and after CsA treatment.246-247 Dummer et al have observed high incidence of herpes simplex virus infection in CsA treated heart transplant recepients.248 Animal studies have shown that infection with viruses such as Sindbis virus, vaccinia249 and herpes virus250 are worsened after CsA therapy. CsA enhanced the release of vesicular stomatitis virus and polio in murine cell line in one study.251 Similarly, other viral infections such as herpes zoster and molluscum contagiosum occur floridly in HIV infection.56

CsA has also been shown to induce a number of bacterial infections including pneumocystis carinii in rats,252 while infections such as florid mycobacterium tuberculosis do not occur as frequently in CsA treated patients as in AIDS. This could be related to the degree of immunosuppression which is not as great with therapeutic dose of CsA as seen in HIV infection. Takashima T. et al showed in mice experiment that dose dependent inhibition of resistance to the BCG strain of Mycobacterium bovis is seen during cyclosporin treatment.253 Among the various fungi, CsA has shown to significantly increase virulence of cryptococcus in rabbit model.254 Extrapulmonary cryptococcosis is common in AIDS56 but the matter is complicated as CsA itself has an anti- fungal activity.255

AUTOIMMUNITY: Although CsA has been used clinically to control allograft rejection, GVHD and some autoimmune diseases,256 it paradoxically induces syngeneic GVHD and other autoimmune diseases in some cases.257 The probable mechanisms include blocking of intrathymic clonal deletion of autoreactive T-Cells,258 deletion of peripheral T-Cells and inhibition of anergy induction48 and inhibition of “veto cell” mediated clonal deletion of post thymic precursor cytotoxic T lymphocytes.259 Similarly, autoimmune phenomena with similarity to SLE260and GVHD261 occur in HIV-AIDS. Antibodies to DNA, erythrocytes, lymphocytes, neutrophils, platelets and immunoglobins are common.262-264Probable mechanisms of HIV induced autoimmunity include viral induction of polyclonal B cell activation, coating of infected cells with viral proteins and making them targets for cytotoxic T cells, increased MHC-II expression of immune and epithelial cells and cross reactions between some viral antigens and MHC-II molecules.265

MALIGNANCIES: After any form of profound and prolonged immunosuppression, malignancies do arise.266 It has been seen that although the incidence of malignancies following the use of CsA is no higher than that with conventional immunosuppressive therapy (CIT), the type and clinical pattern is different and closely resembles that with HIV infection.

Kaposi’s Sarcoma is the most common neoplasm in patients with AIDS267; similarly, KS is very common following CsA treatment and its incidence is 2-4 times higher than in patients receiving CIT.268 Moreover, in CsA treated patients, the onset of the disease is much earlier than in CIT.268 Epidemiological evidence strongly suggests that both AIDS associated and non AIDS associated forms of KS have infectious causes.269 Earlier, CMV was the only pathogen which was commonly found to be associated with KS .270 Chang et al (1994) recently identified DNA sequences of a new herpes virus, provisionally termed KS associated herpes virus (KSHV) in vast majority of AIDS associated KS lesions.271 Similar sequences have now been identified in post transplantation KS.272 HIV tat may also act as a cofactor in induction of KS. HIVtat has shown growth promoting effects on KS spindle cell cultures in vitro273 and tat receptors are present in HIV-1 associated KS.274 CsA associated KS regresses following withdrawal of CsA treatment,275-276 suggesting, that CsA probably has a direct promoting action on KS cells.277 It has also been seen recently that HIV-tat down regulates the expression of P53 oncosuppressor gene product resulting in AIDS related malignancies.278 Similar downregulation of P53 has been seen in transplanted, CsA treated, KS patients. 279

Lymphomas: B-cell lymphomas are common in AIDS246 and post transplant CsA therapy.268 Gastrointestinal tract is the most common extranodal site for lymphomas in HIV infected and post transplant CsA treated patients.247,268 EBV has been implicated in both CsA and HIV associated lymphomas.247-248 It is believed that the virus gets activated after T-cell inhibition, causing B-cell proliferation which may lead to the emergence of a neoplastic population of B-cells.247Another mechanism is that viral tat can also activate B-cells280 leading to their hyperproliferation. Similar direct growth promoting effect on lymphomas has been seen with CsA.275 Other malignancies common in AIDS including cervical carcinoma and anal carcinoma are also common with CsA therapy.268,281

It is evident from the above review that both CsA and HIV share common pathophysiological pathways in achieving the common goal of immunosuppression. CsA achieves immunosuppression by specifically targeting IL-2 genome, while HIV targets cell genome and hijacks its functional capabilities. HIV and CsA also have structural and functional similarities; gag and CsA have b -barrel structure that binds to similar intracellular targets (cyclophilins), tat and CsA augument TGF-b secretion and fibrosis, downregulate P53 gene and promote tumour growth and,tat, gp-120 and CsA induce apoptosis in CD4 cell lines. Interestingly, other pathological effects like organ system abnormalities, cacinogenesis, infections, autoimmunity, angiogenesis and destruction of immature thymocytes also show great simulation. HIV,being a progressive infection, produces the above pathological changes in a quantum and time dependent manner, analogus to the dose dependency seen with CsA, which has been further supported by toxicological studies done in animals. However, regulated therapeutic use of CsA causes minimal and tolerable side effects.

Homoeopathic philosophy, Similia similibus curenter, suggests that when a toxic substance is given in dilutions ( potentised form ), it brings cure for the toxic effects it produces when given in crude form. Keeping this in mind, we prepared various dilutions of Cyclosporin.

PRELIMINARY STUDIES WITH POTENTISED CYCLOSPORIN (Homoeopathic form)

1. Three potencies of the drug, 30,200 and 1000 and a control, were tested in normal Albino mice (ten animals in each group) to see their effect on various organs. The drugs were given orally, once a day, for three months. No change was observed in the behavior, weight and food intake. Autopsy did not reveal any gross abnormality in liver, kidney, thymus, spleen and lymph nodes (routine histology).

2. Lymphocytes from mice sensitised with Salmonella antigens (live or inactivated), when incubated with this drug showed increase in IP3, intracellular Ca2+, increased PKC, increased production of IL2 & gamma IFN (Thl) and increased production of IL4 (Th2) and increased TNF- a . These observations indicate that even Homoeopathic drugs can be tested in vitro.

3. When lymphocytes from two HIV-1 infected patients (CD4 count 400 to 500) were incubated with this drug, the following observations were made:

MNC      48 ± 90  (Background counts)
MNC + PHA   47,577 + 5,324 (Positive control)

MNC + Control Compound 

41,117 + 324 (Control for test compound)
MNC + Test Compound(30)    47,039 + 2,943
MNC + Test Compound (200)

49,423 + 5,765

  To us these results indicate that this drug has potential to stimulate lymphocytes. What is to be seen further is, how in the presence of this drug, lymphocytes react to HIV, whether this drug reduces infectivity, viral load or prevents apoptosis. The present antiviral drugs are handicapped as: (1) they develop resistance, (2) have access to a few viral reservoirs and (3) are not free from side effects. This drug acts on the host and it may be possible that training of the host to fight virus is a better alternative than antiviral drugs(s) alone.

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Acknowledgement-We are highly thankful to Dr Sudhir Gupta,USA and Dr N K Ganguly, Professor and Head, Department of Experimental Medicine, PGI, Chandigarh, India for the laboratory support.

Address for correspondence: Dr NK Mathur, C–24 Peeyush Path, Bapu Nagar, Jaipur – 302015, INDIA

E- mail nkmathur@nkmathur.com

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