Cytokine modulators regulate inflammatory responses: new drug from old African plant
 

Gorilla Staple Adds Spice to New Drugs

By Cheryl Lyn Dybas
Special to The Washington Post
Monday, November 27, 2006; A08


NEW BRUNSWICK, N.J. -- A clear vial filled with amber fluid rests on scientist Ilya Raskin's desk, glinting in the autumn sunlight streaming through his office window. The container, a small glass bottle with a plain white screw-top, contains a substance Raskin calls 006. "Double-zero-six" is potentially more precious than the rarest topaz.

Raskin is a biochemist at Rutgers University's Biotechnology Center. The golden liquid on his desk may prove to be one of the most powerful anti-inflammatory substances ever discovered. "It contains a derivative of a plant known as grains of paradise, or Aframomum melegueta, a member of the ginger family," said Raskin. The compound works in a similar way to the well-known anti-inflammatory drugs Vioxx, Celebrex and Bextra but, it is hoped, without their side effects, said Raskin and other scientists.

Aframomum is not easy to come by. It grows in just one place: the vine-choked swampy lowlands of West Africa's Grain Coast. Stretching from Sherbro Island in Sierra Leone to Cape Palmas in Liberia, this rain-drenched, humid land is named for its abundant grains of paradise.

Outside Africa, Aframomum is usually available only as a hard-to-find spice. For their experiments, Raskin and colleagues hire African botanists to inspect the seeds and ship them to the United States.

Raskin first became interested in Aframomum during an international effort to search for medicines from plants. "Aframomum contains compounds called gingerols, which are chemically similar to other anti-inflammatory compounds," he said. "That's what initially drew our attention to the plant, and was confirmed in the lab."

Plant-derived drugs are hardly new: aspirin, for example, is a synthetic version of a natural substance found in willow bark, and the heart medication digitalis is made from the foxglove plant.

Humans may not be the only creatures that use Aframomum to treat inflammation and infection, said primatologist Michael Huffman of Kyoto University's Primate Research Institute in Japan. He said studies have shown that Western lowland gorillas in Africa prefer Aframomum shoots and seedpods to other foods.

In zoos, the absence of Aframomum and other African plants in the feed given to captive Western lowland gorillas may be a factor in an unexplained heart condition many have developed, say Ellen Dierenfeld, staff nutritionist of the St. Louis Zoo, and Melissa Remis, a primatologist at Purdue University.

"Western lowland gorillas in captivity aren't fed African plants," Dierenfeld said. "We need to look very closely at this aspect of their health to see if there's a link among diet, inflammation or infection, and heart disease."

For humans afflicted with inflammatory diseases, scientists are taking their cue from native African healers, who have used Aframomum for centuries to treat infections of all kinds, said biochemist Christopher Okunji of the National Institute of Allergy and Infectious Diseases.

"In the West African culture in which I was raised," Okunji said, "Aframomum is an important part of daily life. For example, when a visitor arrives at someone's home, no discussion begins until all partake of Aframomum seeds. People far back in African history likely knew that Aframomum was a good thing to eat if you didn't want to get sick."

Okunji's research has shown that one species of Aframomum has significant antimicrobial activity in laboratory tests. In a published study involving cell cultures, Okunji showed that the plant works against the microbe responsible for a hard-to-treat infection, methicillin-resistant Staphylococcus aureus. MRSA, which has reached epidemic levels in some hospitals and other confined places, is impervious to every penicillin-like antibiotic now available.

Okunji has conducted research at Raskin's lab, where lucky visitors may leave with their own thimble-size jar of bronze liquid and a pinch of Aframomum seeds, from which "006" is derived.

"If you spread a thin layer of this substance on a paper-cut or an aching joint," said scientist Neb Ilic of Phytomedics Inc., a pharmaceutical company in Jamesburg, N.J., "there's a warm sensation for a brief time, then the inflammation disappears."

Ilic is a visiting scientist at Rutgers, which has patents pending on Aframomum-related discoveries. Rutgers has licensed those rights to Phytomedics, Raskin said.

Phytomedics has licensed cosmetic-only rights to Avon Products Inc. to manufacture skin-care products that contain Aframomum, said Tolo Fridlender, president of Phytomedics.

Avon scientist Xiaochun Luo said the cosmetic's development is based on what Luo calls Aframomum's superior ability to counteract skin irritation. Avon expects to release the products next spring or summer.

Phytomedics is not alone in its quest to market Aframomum. Another group of researchers, headed by Kenneth Kornman, president and chief scientific officer of Interleukin Genetics Inc., in Waltham, Mass., also has a patent pending on Aframomum applications. Kornman and partners in Interleukin Genetics conducted a clinical trial in humans, completed last summer, of Aframomum's ability to inhibit a component of the immune system known as cytokine modulators.

Cytokine modulators regulate inflammatory responses, which Interleukin Genetics attempted to slow down in its clinical trial. Based on early results, which the company is just beginning to review, said Kornman, "Aframomum might successfully be used to treat diseases with inflammation as their hallmarks, like cardiovascular conditions, arthritis, osteoporosis and Alzheimer's disease."

The clinical trial included blood tests for markers of inflammation, such as C-reactive protein, in volunteers treated with Aframomum or substances from other plants -- blueberry, blackberry and rose hips.

"Although it's too early to say for sure which plant had the most effect, inflammatory markers in people in the Aframomum group responded differently," said Kornman.

In earlier tests in cell cultures, Aframomum "at a very low concentration significantly inhibited the production of C-reactive protein," he said.

If Aframomum lives up to the current hopes for it, Okunji said, "we will owe a great debt to early native healers in Africa" -- and the wild lowland gorillas whose habits they perhaps observed and mimicked.

Wow!
 

Very kool Michael

I just happen to get Avon books every month and order sometimes my bud is a distributer here.. that shows wonderful promise thank you so much for posting that.. I am gonna email that to some friends who need hope too.

hugs an stay warm,
Sandra

Hey there Sandra -

The author of the Washington Post piece is participating on an on-line furum at Noon Eastern Time on Moday, November 27th. Questions can be posted for her in advance at:

http://www.washingtonpost.com/wp-dyn...112400432.html

Mike

Mike as unsual another Great Article from You.

I thought I would add this as well. I have no medical background so please check with your MD. Hugs, Roz


http://www.wipo.int/pctdb/en/wo.jsp?KEY=05/25586.050506

Michael, thank you for the cytokine info that you provided. I have been following your saga about wanting to have yours tested and half a year ago took in one of the explanations of rsd and cytokines to a few of my specialists dealing with my various diagnoses. All of them did show interest by taking the time to read it in front of me.

Thanks, for your updates on what your own investigations about this.
Ina

Dear Ina -

Thanks for your kind note. I wish I could suggest that faster progress was being made in this area. However, I just came across a new study "Changes in immune and glial markers in the CSF of patients with Complex Regional Pain Syndrome," Guillermo M. Alexander, Marielle J. Perreault, Erin R. Reichenberger and Robert J. Schwartzman Brain Behav Immun. 2006 Nov 25; [Epub ahead of print], which suggests a somewhat less robust role for the role of IL6 (60% of all tested CRPS patients had elevated levels) than had been suggested by the authors' prior study, G.M. Alexander, M.A. van Rijn, J.J. van Hilten, M.J. Perreault and RJ. Schwartzman, "Changes in cerebrospinal fluid levels of pro-inflammatory cytokines in CRPS," Pain 116 (2005), pp. 213–219.

The "Discussion" portion of the most recent article - except for a couple of charts that wouldn't copy - follows:

The pathophysiology of CRPS is not well understood, but the evidence indicates that it includes different biological pathways involved in inflammation and central processing of afferent input. Inflammation, tissue damage or nerve lesions can lead to hypersensitivity and allodynia at the site of injury. In some individuals, the pain persists long after the initiating event has healed. There are a number of mechanisms that attempt to explain the pathophysiology of these chronic pain states. Some of these mechanisms are centered on neuronal sensitization (Ikeda et al., 2006 and Woolf and Salter, 2000) and others on neuroimmune interactions and the activation of glial cells (DeLeo and Yezierski, 2001, Marchand et al., 2005, Tsuda et al., 2005 and Watkins and Maier, 2005).

Studies in animal models of exaggerated pain demonstrate that following tissue injury or inflammation, glial cells (astrocytes and microglia) become activated (Tsuda et al., 2005). Some of the signaling molecules implicated in glial activation include; MCP1, fractalkine, ATP, pro-inflammatory cytokines, substance P and glutamate (Abbadie, 2005, Abbadie et al., 2003, Inoue, 2006, Klein et al., 1997, Nakajima and Kohsaka, 2001, Nishiyori et al., 1998 and Svensson et al., 2003). Once activated, microglia and astrocytes secrete a number of substances known to excite dorsal horn neurons and influence the establishment and maintenance of neuropathic pain (Watkins and Maier, 2000). These substances include pro-inflammatory cytokines, nitric oxide, excitatory amino acids, prostaglandins and ATP (Abbadie, 2005, Marchand et al., 2005 and Wieseler-Frank et al., 2004).

This study set out to investigate changes in levels of multiple biological markers in the CSF of individuals afflicted with CRPS and in patients suffering with other non-painful or painful conditions. The use of markers combined with the clinical examination is essential in determining the presence or absence of disease and monitoring its response to therapy.

CRPS patients in this study demonstrated elevated CSF levels of both glutamate and calcium when compared to normal individuals. The release of glutamate and substance P in the spinal cord dorsal horn following inflammation or tissue injury (Hunt and Mantyh, 2001 and Willis, 2001) can potentially lead to calcium dependent long term potentiation resulting in hyperalgesia (Ikeda et al., 2006). Increased extra cellular calcium has been shown to activate nitric oxide synthase (NOS), which is required for the maintenance of hyperalgesia in animal models of persistent pain (Meller and Gebhart, 1993). In addition, variations in extracellular calcium have also been shown to affect neurotransmitter quantal size as well as the probability of transmitter release at central synapses. This effect is mediated by group I metabotropic glutamate receptors (Hardingham et al., 2006).

We evaluated CSF levels of the pro-inflammatory cytokine IL-6, in order to extend our previous work showing an elevation in CSF IL-6 in many CRPS patients. The control patients for CSF IL-6 levels were individuals with radiculopathies, peripheral neuropathies, and NPH. These patients demonstrated CSF IL-6 levels (n = 18, 1.32 pg/ml) similar to previously reported values in normal human volunteers (n = 24, 1.27 pg/ml) (Steensberg et al., 2006). In agreement with our previous work, the CRPS patients in this study showed significantly (F(1, 20) = 8.59, p < 0.01) elevated levels of CSF IL-6 as compared to the control group. The increase in CSF IL-6 was not universal, and was only seen in approximately 60% of the CRPS patients in this study. It is also not specific, as patients with other conditions such as Alzheimer’s and Parkinson’s disease (Blum-Degen et al., 1995), seizures (Lehtimaki et al., 2004), sepsis (Verboon-Maciolek et al., 2006) and spondylolisthesis (this study) show significant increases in CSF IL-6 levels. However, our data do show that the elevation of CSF IL-6 in CRPS is greater than that seen in Alzheimer’s and de novo Parkinson’s disease where neuroinflammation has been proposed as part of the neurodegerative process (Blum-Degen et al., 1995).

With the exception of the NPH group, the CSF levels of the chemokine IL-8 were elevated in all of the patient groups in this study as compared to published values for normal control volunteers (15.5 pg/ml) (Natelson et al., 2005). The values for IL-8 in the CSF of the CRPS, radiculopathies, peripheral neuropathies, spondylolisthesis and ALS patients were comparable to levels reported in patients with postherpetic neuralgia (PHN) (35 pg/ml) (Kotani et al., 2000). In patients afflicted with PHN, the increase in CSF IL-8 correlates with both the degree of pain and the duration of disease (Kotani et al., 2000). In this study, IL-8 levels did not correlate (r(1, 49) = 0.06, p = 0.68) with pain levels (VAS scores) and significant differences were not noted (F(1, 49) = 0.29, p = 0.59) in CSF IL-8 levels between patients reporting chronic pain (n = 39, 39.7 pg/ml) and patients reporting no pain (n = 12, 33.7 pg/ml).

In this study, the patients in the ALS group demonstrated significantly greater (F(1, 48) = 7.4, p < 0.01) CSF levels of MCP1 as compared to all other patient groups. The CSF levels of MCP1 in the ALS patients in our study (518 pg/ml) are comparable to previously reported values for MCP1 in ALS patients (570 pg/ml) (Wilms et al., 2003). In their study, the CSF level of MCP1 in their control group (tension headaches) was 285 pg/ml, which is much less that the CSF level of MCP1 in all of the groups in our study, suggesting that all of the groups in this study, including the CRPS patients, demonstrate elevated CSF levels of MCP1.

Following injury or inflammation, MCP1 is expressed by both neurons (Zhang and De Koninck, 2006) and glial cells (Babcock et al., 2003). The major source of MCP1 expression comes from microglia and GFAP positive astrocytes (Babcock et al., 2003). GFAP, a protein member of the intermediate filament family, is strongly expressed in activated astrocytes and its level in CSF was also increased in all patient groups in this study when compared to published values for age-matched neurologically healthy individuals (Rosengren et al., 1994 and Anderson et al., 2003). Given that activated astrocytes are the source for GFAP and one of the major sources of MCP1, it is not surprising that they were positively correlated in the CSF of all patient groups (r(1, 47) = 0.45, p < 0.01), and especially in the CRPS group (r(1, 20) = 0.55, p < 0.01) (Fig. 1).

[Not shown] Fig. 1. Relationship between levels of the chemokine MCP1 (pg/ml) and the protein GFAP (ng/ml) in the cerebrospinal fluid of patients afflicted with CRPS. MCP1 and GFAP were positively correlated (r(1, 20) = 0.55, p < 0.01).

Most of the patients in this study showed elevated levels of nitrate plus nitrite with the patients in the CRPS group demonstrating the greatest elevation. In the CNS, IL-4 and IL-10 are expressed in reactive astrocytes and activated microglia (Hulshof et al., 2002 and Park et al., 2005). IL-4 and IL-10 have been shown to inhibit inducible NOS (iNOS) expression resulting in decreased NO synthesis by glial cells (Koeberele et al., 2004). A reduction of iNOS by IL-4 or IL-10 should result in an inverse correlation between their CSF levels and NO metabolites. There was no correlation between CSF NO metabolites and IL-10 in any of the patient groups (r(1, 42) = 0.06, p = 0.71). However, with the exception of the CRPS patients the CSF levels of NO metabolites (nitrate plus nitrite) in the study patients were inversely correlated with their CSF IL-4 levels (r(1, 14) = 0.60, p < 0.01) in contrast to the CRPS patients which did not show such a correlation (r(1, 11) = 0.32, p = 0.29) (Fig. 2). The lack of correlation between IL-4 and NO metabolites in CRPS patients may be due to the fact that IL-4 levels were not high enough to inhibit iNOS or that NO production in these patients resulted from the induction of other isoforms of NOS. It has been proposed that IL-4 regulates brain inflammation by inducing the death of activated microglia, (Park et al., 2005). The level of IL-4 expression in the CRPS patients may be insufficient to reduce brain inflammation and may be a contributing factor to the mechanisms responsible for the pathophysiology of CRPS.

[Not shown] Fig. 2. Relationship between CSF levels of the NO metabolites (nitrate plus nitrite) (uM) and IL-4 (pg/ml). All of the included samples had CSF IL-4 levels greater than the sensitivity of the ELISA (0.1 pg/ml). In the NPH, radiculopathies, peripheral neuropathies and spondylolisthesis patients (Controls ), NO metabolites were inversely correlated with CSF IL-4 levels (r(1, 14) = 0.60, p < 0.01). In the CRPS (•) patients, there was no correlation between CSF levels of NO metabolites and CSF IL-4 levels (r(1, 11) = 0.32, p = 0.29).

As a group, the CRPS patients in this study demonstrated elevated CSF levels of IL-6, IL-8, MCP1, GFAP, NO metabolites, glutamate and calcium. It was difficult to establish whether the CRPS patients demonstrated elevated or reduced levels of IL-10 and IL-4 as compared to individuals without neurological diseases, since normative values for these cytokines in the CSF range widely in the literature (Bartosik-Psujek and Stelmasiak, 2005, Natelson et al., 2005, Rota et al., 2006 and Stoeck et al., 2005). However, except for the IL-4 levels in the ALS group, the CSF levels of IL-10 and IL-4 in the CRPS patients were the lowest of all of the other disease groups.

There was no elevation or reduction of a CSF marker that was specific to the CRPS patients. However there were several patterns of markers that could be helpful in both elucidating the mechanisms involved in the disease process and supporting the diagnosis of CRPS. The most common pattern was found in 50% (11 out of 22) of the CRPS patients and consisted of; elevated IL-6, low levels of either IL-4 or IL-10, increased GFAP or MCP1 and increases in at least two of the following markers NO metabolites, calcium or glutamate. The second most common pattern was found in 18% (4 out of 22) of the CRPS patients and it consisted of; normal IL-6, low levels of either IL-4 or IL-10, increased GFAP or MCP1 and increases in at least two of the following markers NO metabolites, calcium or glutamate. A third pattern was found in 14% (3 out of 22) of the CRPS patients and it consisted of; increased GFAP or MCP1 and low levels of either IL-4 or IL-10. The remaining CRPS patients showed normal levels of GFAP and MCP1 and demonstrated at least one of the following; elevated IL-6, low levels of the anti-inflammatory cytokines or increases in either NO metabolites, calcium or glutamate.

There were several limitations of this study: (1) We studied a relatively small number of CRPS patients, and a larger sample is needed in order to determine which patterns are relevant to the pathophysiology of the disease; (2) We did not have CSF samples from normal control volunteers and in many cases had to compare CSF levels to previously reported values in healthy individuals. Using values from other studies is limited by the variability seen in the literature for levels of cytokines and chemokines in CSF. However, much of the variability is due to the lack of sensitivity of the methods employed. In this study, we made comparisons of our data to normative values from other studies that used methods with the highest sensitivity available and when possible from the same manufacturer as the ELISA kits we employed; (3) None of the CSF samples were from patients with early CRPS (less than 6 months); (4) We did not have CSF samples from the same individual at different time points in order to match CSF marker patterns with the severity of their symptoms and; (5) More sensitive assays are needed. There were a number of markers (IL-1β, TNF-α and fractalkine) that may have provided additional information, but in most samples their CSF levels were below the level of detection of the available assays.

Our hope is that the data obtained from this and other similar studies may aid in elucidating the mechanisms involved in the pathophysiology of CRPS. A better understanding of these mechanisms may lead to novel treatments for this very severe, life-altering illness. [Emphasis added.]

I would be happy to email a copy of the full article to anyone who wants it. Just send me a pm with your email address: the file is just slightly too large to attach here. (Personal, non-commercial use only, please.)

So now maybe have a better understanding as to why the allergist/clinical immulogist who saw me at Hopkins a couple of months ago wasn't as bowled over by "Changes in cerebrospinal fluid levels of pro-inflammatory cytokines in CRPS," Pain 116 (2005), pp. 213–219, as I had been walking in there. Call it a work in progress.

Mike

Just Updating

Thanks Michael,

I need a bit more time to read that and properly digest it ....

I do recall that one of my specialists did say there are some comments re cytokines that sit on either side of the fence, depending which time one hears about them, but still i know interest was there...

Thanks,
Ina