examinations were done according to the doctors order. Side effects, abnormal laboratory values, and efficacy were specifically recorded at each visit.

Laboratorv tests

Blood samples were collected with blood collection tubes (Vacutainer CPT; Becton Dickinson, Basel, Switzerland). Plasma was separated within 2 hours and either processed immediately or frozen at -80 C for later use. Human immunodefficiency virus RNA viremia was measured using commercially available quantitative polymerase chain reaction equipment (Amplicor mv Monitor; Roche Diagnostics, Basel, Switzerland). The lower limit of detection of this assay is 400 copies per milliliter, and the ultrasensitive method is used for detection ofmv RNA levels between 50-50000 copies

per milliliter. CD4 cell counts were measured using flow cytometry.

Studv end ~oints

Changes in plasma mv RNA concentrations and CD4 lymphocyte counts during therapy were analyzed. II) accordance with British mv Association Guidelines Co- ordinating Committee (1997) and Casado et ai, 1998, we defmed virological efficacy as a reduction in mv RNA by at least 0.5 loglo copies per milliliter; immunological efficacy was defmed as an increase in the CD4 cell count by at least 100 x 106/1. The study is approved by the Ethical committee of the Ministry of Public Health of Thailand.

Table 2 Baseline characteristics of the study patients

Age (yr)/ weight Performance x 106/ 1 (copies/ml) infection gender (kg) Score (yr)

0 ,--Q,,- A1/26/F 61 100 heterosexual 440 45,393 4/12

A2/35/M 67 100 heterosexual 170 30,757 4+4/12

A3/29/F 57 100 heterosexual 339 9,527 10/12

~/41/F 50 100 heterosexual 16 52,800 7/12

As/29/F 60 100 heterosexual 564 31,305 4/12

B1/30/F 48 100 heterosexual 54 119,032 4

B2/24/F 47 100 heterosexual 799 40,160 5

B3/38/F 54 100 heterosexual 531 332,455 1+4/12

BJ29/F 55 100 heterosexual 660 3,038 6/12

Bs/49/M 66 100 homosexual 530 385,298 6

C1/51/M 65 100 homosexual 324 132,000 9

C2/33/M 57 100 heterosexual 80 60,600 2

C3/41/F 50 100 heterosexual 16 52,800 7/12

C4/30/F 42 100 heterosexual 2 118,000 2

Cs/36/M 58 100 heterosexaul 278 9,730 3

-J --

RESULTS

Laboratory

Virological and immunological findings of all patients are presented in Fig 1,2

and Fig 3,4 respectively. The outstanding case C3 had a reduction oh mv RNA from 52,800 to 55 copies/ml or from 4.72 to 1.75 log RNA copies/mI. Biochemical findings were that there was no side-effect on the livers or kidneys of the 15 patients.

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Clinical

The characteristics of all 15 patients are listed in Table 2. There were 5 men and 10 women, ages ranging from 24 to 51 years. All patients had an increase in appetite and gained weight ( 2 to 10 kg) while taking  plymate formulation. No patient contracted long-time fever. When a patient had short-time fever, they were able to recover by the immunity in their bodies, and no analgesic agent was required. No patient contracted pruritis due to allergy to mosquito bite while taking  plymate formulation. Case C3 who bad a reduction of mv viral load from 52,800 to 55 copies/ml,she felt pain in her bre8$t after starting to take  plymate formulation for 2-3 days and then she felt fresh, was hungry frequently and gained weight 10 kg in 2 months.Before taking  plymate formulation she had eruption all body. After treatment for 2 months,all eruption disappeared. She had tuberculosis before taking  plymate formulation,after taking  plymate formulation for I month,she was not suffer from tuberculosis any more. Case Bs experienced flattening in 2 cervical lymph node enlargements, and an apparent incease in musculature while taking  plymate formulation; short-sight eyeglasses did not need to be worn because of myasthenia gravis.

MDK 2000 had no observed effect on the livers or the kidneys of any patient while taking the drug.

DISCUSSION

Laboratory

Virological fmdings

Most patient mv RNA curves decreased after 3-4 weeks of  plymate formulation A2000 therapy, which is an indication of the suppressive action of  plymate formulation. After that, the curves tended to increase and then decrease again. These fmdings suggest that some mv- infected macrophages are slowly released from the lymph nodes, as we found a decrease in the size of patient lymph nodes (Bs). Macrophages are major reservoirs for mv during all stages of infection. Unlike the lytic infection of T cells, mV-infected macrophages persist in tissue for extended periods (months) with large numbers of infectious particles contained within intracytoplasmic vacuoles (Meltzer, et ai, 1990). After the increase phase, there are repeated decreases in mv RNA. One possible explanation for this may be as follows: since the methods for measuring mv RNA viremia are imperfect, because of the inability to distinguish live viruses from dead viruses that may come from mv -infected macro phages that persist in tissue for a long time. After two patients (As and Bs) stopped taking  plymate formulation for 3-4 months (during weeks] 2-22, see Fig. ]), it was found that mv RNA decreased despite taking nothing during the period. Since there is impaired antibody-dependent cell-mediated cytotoxic activity in patients with acquired immunodeficiency syndrome, during this 3-4 months of stopping plymate formulation therapy, the repaired immune system, probably because of MEDS

I2000, could completely clear all dead viruses.For Case C3,she may receive specific antibody of HIV type which match with my in her body from plymate formulation.

i Immunological findings

Several cohort studies have demonstrated that CD4 lymphocyte counts decrease with the duration of my infection (Moss, et aI 1988). This decrease tends to be linear

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and predicts opportunistic infection and progression to acquired immunodeficiency syndrome (AIDS) (Moss, et aI1988). However, there are limitations in using CD4 counts to evaluate the stages of lIIV infection in patients, since CD4 lymphocyte counts are an imperfect measurement of the duration of lIIV infection. In addition. there is considerable variability in the normal level of CD4 counts in uninfected persons (Malone, et a11990) and in the rate of decline of CD4 counts among lIIV -infected persons (Phillips, et al 1989). Therefore, the changes in CD4 counts of our patients were not able to show any response clearly, except for long-term responses that were clearly demonstrated by increasing CD4 counts of three patients (As, B4 and Bs) during weeks 24-29.

Clinical

Lymph node enlargement

Infection with lIIV is characterized by progressive and profound deterioration of the host immune system with a long period of clinical latency (Fauci, 1988). The immune system in the latent phase is largely intact, but there is low level lIIV replication, predominantly in the lymphoid tissues, which may last for several years. Five to twenty percent of patients with lymphadenopathy proceed to develop full-blown AIDS after a mean observation time of 12 to 22 months (Rashbeing-Belcher, et aI1986). In addition, there is evidence that certain benign histological entities can progress to malignancy. It has been suggested that atypical lymph node hyperplasia may progress to malignant lymphoma (Said, 1988). Burns et a/ (1985) believe that all patients with lymphadenopathy should undergo biopsy (Gerstof, et aI, 1989). Wong et a/ (1991) found that 64% of lymph node biopsies in patients infected with lIIV resulted in the institution or alteration of treatment. Some treatment modalities can be curative and may improve survival in patients who might otherwise die of the disease. This suggests that enlarged lymph nodes in lIlY-infected patients can lead to greater problems later. Our findings that Bs had a decrease in size of one enlarged cervical lymph node and flattening of another while taking MDK 2000, is a good sign of recovery of immune deterioration. Harrer et a/ (1999) found that the parameters distinguishing lIlY-infected homosexual males with lymphadenopathy syndrome ftom those without progression of disease were higher serum levels of gamma-interferon. higher relative and absolute numbers of CDs- suppressor cells in the peripheral blood, and a reduced number of CD4-helper cells in the lymph nodes.

Pro ritis

Pruritic papular eruption (PPE) is the most common cutaneous manifestation in lIlY-infected patients. All of the patients had PPE because of mosquito bites before taking A2000. PPE has been suggested to be due to host immunity (Mllazza, et aI, 1999). None of the patients (100 %) had PPE due to mosquito bites while taking A2000. This may reflect improvement in host immunity, which is attributable to

A2000

Fever

Fever is a ftequent symptom in people infected with lIIV (Follansbee, et aI, 1982)" Fever may be associated with opportunistic infections (Follansbee, et aI, 1982) or with

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bacterial infections due to Streptococcus pneumoniae, Staphylococcus aureus, or Haemophilus injluenzae (Follansbee, et aI, 1982). Findings that our patients did not get long-time fever while taking drplym and could recover from short-time fever without taking any analgesics, may suggest that our patients recovered to some degree from immune deterioration because of my infection.

Appetite

Intact taste and smell function is important in my -infect,ed individuals because chemosensory abnormalities may contribute to inadequate food intake leading to malnutrition, weight loss, and ultimately wasting. Caloric intake and subsequent nutritional status may be affected by impaired taste and smell function (Schiffman, 1983). There are reports of taste and smell dysfunction in the mY-infected population (Brody, et aI, 1998). The patients in this study may have recovered from taste and smell dysfunction, and have an increase in appetite, resulted in weight gain of at least 2 kg.

Muscle

The profound loss of body weight, or wasting, associated with my infection is

widespread and dramatic (Kotler, et aI, 1985), so that it is considered evidenceof the progression of infection to AIDS (Anonymous, 1987). This loss of lean body mass, or body muscle mass in AIDS wasting has grave consequences and is associated not only with decreased survival (Cutler, et aI, 1989) but also with increased hospitalization and diminished quality of life (Turner, et aI, 1994). Although lack of nutrient intake may exacerbate the loss of muscle mass, the problem is not solely one of undernutrition; McNurlan et a (1997) have demonstrated that it is associated with increased basal muscle protein degradation and decreased responsiveness of muscle protein synthesis to growth hormone in the later stages of disease. BS was the patient infected with my for the longest time (6 yr), had developed weakness of the muscles, and became easily fatigued. Afterdrplym therapy, there was apparent increased muscular strength and muscle mass appeared clearly throughout the body; the patient was bale to perform physical exertion to a greater degree than before therapy. This may be due to decreased basal muscle protein degradation and increased responsiveness of muscle protein synthesis to growth hormone because of drplym.

Myasthenia gravis

Skeletal muscle involvement may occur at all stages of my infection and represents the first manifestation of the disease in some patients, such as those with myasthenia gravis (Gheeradi, et aI, 1983). Bs had short-sightedness after my infection, which may have been due to weakness of the optical muscle because of myasthenia gravis. While taking drplym, Bs did not need to wear short-sight eyeglasses.

Proposed action of A2000 in DIV infectiQQ

Lactoferrin is a mammalian iron-binding glycoprotein present in many biological secretions, such as milk, tears, semen and plasma, and a major component of the specific granules of polymorphonuclear leucocytes. Both my -1 replication and syncytium formation were efficiently inhibited, in a dose-dependent manner, by lactoferrins. Bovine

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7

lactoferrin in iron-free (apo) and iron-saturated forms markedly inhibited IllV-l replication when added prior to IllV infection during the virus adsorption step, thus suggesting a mechanism of action on the IllV binding to, or entry into, cells (Puddu, et aI, 1998). The levels of plasma lactoferrin are decreased in IllV-l infected patients in relation to the progression of the disease. The effect of lactoferrin is of great importance in the nonspecific defenses. The real biological place lacking such a molecule could be one important component of the multifactorial nature of IllV-l infection(Defer,et aI, 1995). Furthermore, antilactoferrin auto-antibodies are associated with IllV infection. The susceptibility. of lactoferrin in bovine colostrum to digestion by trypsin and chymotrypsin has been investigated by Brines and Brock (Brines and Brock, 1983). They found that bovine iron-saturated lactoferrin was more resistant to digestion than apo forms. drplym is acidified and prepared as a ferric-saturated form of bovine colostrum. However, further investigation will be performed by injecting patients with

A2000, as well.

It is known from various in vivo and in vitro studies that bovine and human colostrum possess broad-spectrum antimicrobial action (Dolan, et at, 1989). Resistance of the colostral immunoglobulins to proteolysis in the gastrointestinal tract has been known for more than twenty years ago (Rham and Isliker, 1977). In addition, there is evidence that infection with one pathogen can benefit the host through immunological effects on a second unrelated organism (Colin, et aI, 1999). For example, IllV-l infection can decrease the inflammatory response to hepatitis B in chronic hepatitis, but hepatitis B virus replication increases and the overall effect is deleterious (Bloom, 1998). Scrub typhus was formerly diagnosed by the Weil-Felix test, which detects antibodies that cross-react with a different organism, Proteus mirabilis. This immune cross-reactivity does not benefit the host, but is a precedent for the formation of cross-reactive antibody during scrub typhus infection. Recently, Watt et al (2000) found IllV -1 suppression during acute scrub typhus infection. Therefore, non-specific antibodies in bovine colostrum may suppress IllV infection.

The American Indian herbs may have a positive effect on the treatment of mv- infected patients.

We propose that A2000 inhibits viral replication and attacks IllV -infected macrophages in tissue, such as lymph nodes, results in the slow release of IllV -infected macrophages from the lymph nodes, since we found a decrease in lymph node size in Bs, and since macrophages are major reservoirs for IllV during all stages of infection. Unlike the lytic infection of T cells, III V -infected macrophages show little or no virus- induced cytopathic effects. IllV -infected macro phages persist in tissue for extended periods (months) with large numbers of infectious particles contained within intracytoplasmic vacuoles (Meltzer,et aI, 1990). Therefore, some of the curves of IllV RNA decreased, while some increased because of the slow release of IllV from tissue. The number of HIV RNA that can be measured by this method include live as well as dead viruses. It take a long time (about 3-4 months) after patients stop A2000 therapy before their immune system can completely clear all dead viruses. As a result, after stopping drplym therapy for a period of 3-4 months, the number of HIV RNA decrease despite taking nothing during this period, because there is impaired antibody- dependent cell-mediated cytotoxic activity in patients with acquired immunodeficiency syndrome (Bender, et aI, 1986).

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