Prelude

Since 1997 a number of parents with an autism-spectrum child have requested that
I peruse the child's immune-related lab-test data, often accompanied by other
metabolic information, occasionally by a complete medical history. However, much
credit goes to Hugh Fudenberg, MD, who years ago realized the importance of
immune-shifts and infectious processes in many autism-spectrum children, and who
ordered tests and designed treatments accordingly, for each specific child (1).
     My small sampling's immune data reveal that most of these autism-spectrum
children have atypical elevations of antibodies against otherwise common
pathogens such as Epstein-Barr virus, Cytomegalovirus, and/or Human Herpes Virus
6 (EBV, CMV, HHV6), and in some cases, elevated anti-measles antibodies. 
     However, the subject "what tests to order" in diagnosing autism-spectrum
children is not simple, even as a number of parents -- along with some
physicians and researchers -- are helping create a diagnostic algorithm -- based
upon a growing amount of lab-test data from actual autism-spectrum children
whose parents managed to find doctors and researchers willing to assess and
understand biological factors. 
     The ideas offered in this webpage are presented as neither complete nor
final; much refinement is needed, as is input from others, the ideas on this
webpage are presented on behalf of developing a comprehensive biological
algorithm for diagnostics regarding autism-spectrum children.

The immune data presented hereinbelow (8) were purchased by parents of an
autism-spectrum child and are reproduced with permission of the parents; an
analysis of his specific case shall be presented on a separate page. Hugh
Fudenberg, MD, ordered most of these tests. He looks at a wide range of
immunologically significant factors, including several tests regarding
complement. Importantly, many of the tests generated data about IgG antibodies
and, with some exceptions, did not include assays for IgM antibodies. This is
an important distinction.
     Generally speaking, IgM antibodies reflect a recently acquired infection,
whereas atypical elevations of anti-pathogen IgG represent a chronic infection
not recently acquired, possibly even acquired in utero (eg, 1). With these
IgG/IgM considerations in mind, also important to notice is that Dr. Fudenberg's
panel (and others I've examined) have focused extensively upon EBV, whose
effects and reactivations are complex, thus prompting both IgM and IgG
evaluations for several EBV-related proteins.

The atypically elevated (ie, mid-level elevations) I see in most of the autism-
spectrum kids' immune-panels I've perused suggest that the child (i) has
sufficient overall immunity so as to keep the child from appearing sick, yet
(ii) has an underlying immune-impairment (a) that allows an atypical chronic
infection represented by atypically elevated antibodies, (b) that may be genetic
and/or infectious in origin, and that is indicated not only by the atypical
chronic infections but also by altered CD4 ratios and NK variance regarding NK
count and/or function.
     Furthermore, many of the charts reveal more than one atypically elevated
anti-pathogen antibodies level, suggesting co-infection; and usually these
elevated titres have been directed against viral antigens (2), which is
important because synergistic effects of co-infection have been described (eg,
3-4).

The fact that most of the autism-spectrum immune-panels I've perused contain
either (a) signs of an immune irregularity, and/or (b) atypical elevations of
at least one and *usually several* anti-viral antibodies titres suggests that
a new subgroup in autism-spectrum diagnostics is becoming identified, with the
following characteristics:

  (i)  Atypical elevations of common pathogens -- usually regarding EBV, CMV,
and/or HHV6, occasionally HSV or measles. 
 (ii)  Many of these immune panels not only contain atypically elevated IgG-
antibodies but also delineate a specific immune impairment revealed by the
absence of antibodies against a common antigen for which antibodies ought be
present. Such "missing" or "none detected" antibodies have included varicella,
tetanus, diphteria, etc. 
(iii)  Regarding HSV and CMV, *seemingly* normal peripheral-antibodies levels
of HSV and CMV are difficult to interpret and may represent false-negatives (5),
but an atypical elevation of either is important (6).
 (iv)  Atypical antibodies elevations or missing antibodies are not the only
unusual features of these charts. In some cases, the immune panel reveals other
atypical elevations, eg, mild antibody elevations that (while appearing
significant) *may* reflect adjuvant effects from viruses or other pathogens.
  (v)  Generally -- and there are exceptions -- many of these kids appear to
have an immune shift in a Th2 direction. 

While the initial number of immune panels analyzed remains small (n<15), their
data suggest: (a) that a thorough immune panel ought be ordered for many autism-
spectrum children, (b) that certain treatment protocols (eg, Prednisone) be
enacted only *after* chronic infections have been ruled out, and (c) that
restoring immune-competence and reducing the level of chronic infection are
legitimate parental and medical goals in these children.

I do not suggest that *all* autism-spectrum children will be found to have the
"atypical antibodies profile", far from it; and an important ramification is
financial. If a parent pays for a thorough immune panel, there is no guarantee
that the child's panel will provide useful information, ie, no obvious
atypicalities may emerge. 
     Furthermore, as of 1999, the data provided in these panels is at the
forefront of medical treatment and research. Many docs (i) rely upon the
comforting notion "Well, most kids get these infections" and (ii) know how to
identify a child experiencing an extreme illness from one of the pathogens
increasingly identified as atypical in autism-spectrum immune panels. A major
ramification of this invalid but widely accepted dichotomy among physicians
concerns treatment.

There are anti-virals for several of the viruses which are producing atypical
chronic infection in a subgroup of autism-spectrum children, yet my impression
is that these antivirals usually are not used unless symptoms generated by the
infection are obvious and severe. Thus, when parents obtain a thorough immune
panel that reveals an atypical chronic infection (or several!), the parents may
find their child in yet another medical limbo, in that docs may be habituated
not to use the pharmaceuticals effective against the pathogen with atypical
presence in their child -- eg, EBV, CMV, HHV6, or HSV -- even though these drugs
would help reduce the viral presence in these children. 
     Furthermore, and this is no surprise to many autism-spectrum parents circa
1999, restoring immune competence may be an important component of long-term
success, a point also realized and mentioned by Dr. Luigina Romani in her
Orlando presentation and by Michael Goldberg, MD, in a recent post to the NIDS
list.

My *hunch* as a researcher is that lowering the viral load from viruses like
EBV, CMV, and/or HHV6 ought be helpful, and such efforts should occur in a
larger therapeutic context of re-normalizing the child's immune status. 
     An additional *hunch* is that -- for kids whose immune panels indicate
atypical chronic infection -- low-dose, long-term regimens with specific anti-
virals is preferable to the high-dose, short-term strategies needed for kids
with a raging infection, which these autism-spectrum kids do not have. And,
these efforts -- if and when attempted -- ought occur in the context of
rebuilding the child's immune competence.

For most of the kids whose chart's I've perused, practical treatments seem
within reach; certain atypical chronic infections can be reduced in severity.
However, given the well ingrained physicians' dichotomy of "really sick" or
"these viruses don't matter", parents who identify atypical chronic infection
and want treatments are in the position of reshaping modern medicine. 
     For me, the purporedly "genetics" question "How many engineers are in your
family?" is being replaced by lab-test data and solid science. A more important
question would be, What are the NIH, pharmaceutical companies, and researchers
doing in regard to immunosuppressing atypical chronic infections? 
     Just as healing the gut has helped so many autism-spectrum kids, so too
minimizing atypical chronic infections and restoring immune competence are
worthy goals (eg, 7).

In the autism-spectrum immune panels I've perused, the antiviral antibodies data
have been the most instructive, and the CD4, CD8, CD3, and NK data are also
useful, and the complement data and vaccinal antibodies levels provide important
baselines.
     Importantly, the immune categories listed in the sample chart did not
mention IgE and were very sparse in regards to IgA, thus the sample data-set
presented here is not complete. IgE and IgA profiles are extremely important,
in the context of a child's thorough immune-panel, along with other biological
analyses documented elsewhere. 
     In a subsequent post and webpage immune data specific for the child
mentioned above shall be presented. Comments and suggestions are welcomed. 
Teresa

1. Britt WJ, Vugler LG. Antiviral antibody responses in mothers and their
newborn infants with clinical and subclinical congenital cytomegalovirus
infections. J Infect Dis 1990 Feb;161(2):214-9.
          Department of Pediatrics, School of Medicine, University of
          Alabama, Birmingham 35294.
ab: Human cytomegalovirus (HCMV)-specific antibodies were assayed in cord serum
and the respective maternal serum from two groups of newborn infants with
congenital HCMV infection. One group of infants exhibited clinically apparent
infection with multiple organ system involvement, whereas the second group had
subclinical infections. Levels of virus-specific IgG antibodies reactive with
several virus-encoded proteins including those reactive with the major envelope
glycoprotein complex were significantly higher in cord serum and maternal
delivery serum from the group of infants with clinically apparent infection than
in serum from those with subclinical infection. Maternal delivery serum from the
group with subclinical infection had significantly higher levels of IgM
virus-binding antibody and lower levels of IgG virus-binding antibody than did
the respective serum from the group with clinically apparent infection,
suggesting maternal acquisition occurred later in gestation in the group with
subclinical infection. These results suggested that deficiencies in the
HCMV-specific antibody response were not associated with clinically apparent
congenital infection and that other factors, such as early virus acquisition
during pregnancy, might contribute to the severity of intrauterine HCMV
infection. 
PMID: 2153737, UI: 90131859  

2. I and others are finding mid-level anti-viral antibodies elevations because
parents are ordering the viral-antibodies tests. In any specific autism child,
another pathogen or several -- eg, a fungus, bacteria, or nematode  -- may be
present. These too can shift immunity, and can persist as a chronic, underlying
infection in a host (eg, child) with an underlying immune impairment. For most
kids and in the absence of obvious symptoms of an unusual fungus or bacterium,
etc -- I suggest looking first at the anti-viral antibodies, along with the
vaccination-related antigens, because these viruses are ubiquitous; prenatal,
infant, and childhood exposure is not uncommon; and most children would have
antibodies levels that fall within (yea!, verily determine) the reference
ranges, thus making the atypical elevations significant and noteworthy.

3. Flamand L et al. Activation of the Epstein-Barr virus replicative cycle by
human herpesvirus 6. J Virol 1993 Nov;67(11):6768-77.
          Laboratory of Immunovirology, Ste-Justine Hospital,
          Montreal, Canada.
ab: One common attribute of herpesviruses is the ability to establish latent,
life-long infections. The role of virus-virus interaction in viral reactivation
between or among herpesviruses has not been studied. Preliminary experiments in
our laboratory had indicated that infection of Epstein-Barr virus (EBV)
genome-positive human lymphoid cell lines with human herpesvirus 6 (HHV-6)
results in EBV reactivation in these cells. To further our knowledge of this
complex phenomenon, we investigated the effect of HHV-6 infection on expression
of the viral lytic cycle proteins of EBV. Our results indicate that HHV-6
upregulates, by up to 10-fold, expression of the immediate-early Zebra antigen
and the diffuse and restricted (85 kDa) early antigens (EA-D and EA-R,
respectively) in both EBV producer and nonproducer cell lines (i.e., P3HR1,
Akata, and Raji). Maximal EA-D induction was observed at 72 h post-HHV-6
infection. Furthermore, expression of late EBV gene products, namely, the viral
capsid antigen (125 kDa) and viral membrane glycoprotein gp350, was also
increased in EBV producer cells (P3HR1 and Akata) following infection by HHV-6.
By using dual-color membrane immunofluorescence, it was found that most of the
cells expressing viral membrane glycoprotein gp350 were also positive for HHV-6
antigens, suggesting a direct effect of HHV-6 replication on induction of the
EBV replicative cycle. No expression of late EBV antigens was observed in Raji
cells following infection by HHV-6, implying a lack of functional
complementation between the deleted form of EBV found in Raji cells and the
superinfecting HHV-6. The susceptibility of the cell lines to infection by HHV-6
correlated with increased expression of various EBV proteins in that B95-8
cells, which are not susceptible to HHV-6 infection, did not show an increase
in expression of EBV antigens following treatment with HHV-6. Moreover, UV
light-irradiated or heat-inactivated HHV-6 had no upregulating effect on the
Zebra antigen or EA-D in Raji cells, indicating that infectious virus is
required for the observed effects of HHV-6 on these EBV products. These results
show that HHV-6, another lymphotropic human herpesvirus, can activate EBV
replication and may thus contribute to the pathogenesis of EBV-associated
diseases. 
PMID: 8411380, UI: 94016864

4. Kikuta H, Rapp F. Enhancement of Epstein-Barr virus gene expression by other
herpesviruses. Intervirology 1988;29(5):281-91.
          Department of Microbiology and Immunology, Penn State
          University College of Medicine, Hershey PA 17033.
ab: We examined the effect of infection by other herpesviruses on the induction
of Epstein-Barr virus (EBV) antigens. Raji cells were infected with a variety
of viruses before superinfection with P3HR-1 virus 6 h later. Much earlier and
greater synthesis of EBV antigens was observed in herpesvirus (herpes simplex
virus type 1, type 2 and pseudorabies virus)-infected Raji cells than in
mock-infected Raji cells prior to superinfection with P3HR-1 virus, as
determined by indirect immunofluorescence tests or immunoprecipitation and PAGE.
Although infection with a temperature-sensitive mutant of pseudorabies virus
(tsG1) enhanced EBV antigen synthesis as well as wild-type pseudorabies virus
at permissive temperature in Raji cells superinfected with P3HR-1 virus, EBV
antigen synthesis was strongly inhibited at temperatures nonpermissive for tsG1.
These results suggest that a herpesvirus immediate-early protein may play a role
in enhancement of EBV antigen expression. 
PMID: 2848780, UI: 89066194

5. As mentioned in citation 1, early CMV can be asymptomatic and is associated
with impaired production anti-CMV antibodies, a phenomenon reported earlier:

5a. Pass RF et al. Specific lymphocyte blastogenic responses in children with
cytomegalovirus and herpes simplex virus infections acquired early in infancy.
Infect Immun 1981 Oct;34(1):166-70.
     ab: Cell-mediated immune responses in 27 infants and children with
cytomegalovirus (CMV) infection acquired between birth and 1 year of age were
compared with responses in 13 children who had neonatal herpes simplex virus
(HSV) infection. Infection was asymptomatic in 25 of 27 CMV-infected children;
the 13 patients with HSV infection were all ill as newborns. The median age when
studied was 46 months for children infected with CMV and 24 months for those
infected with HSV. We measured lymphocyte transformation responses (LTRs) to CMV
antigens in the former group and to HSV type 1 (HSV-1) (and in six cases to
HSV-2) in the latter group, with the results expressed as a stimulation index.
Based on the results in seropositive and seronegative adult control subjects,
stimulation indexes of greater than or equal to 3 were considered indicative of
a positive LTR. Among the CMV-infected children, a positive LTR was observed in
0 to 13 assays performed before 1 year of age, 3 of 8 assays performed between
1 and 4 years of age, and 9 of 15 assays performed over 4 years of age. In
contrast, a positive LTR to HSV-1 was seen in 15 to 18 assays performed in
children under 1 year of age and in 14 of 16 assays performed in survivors of
neonatal HSV infection older than 1 year. Six HSV-2-infected patients were
tested simultaneously 13 times with HSV-1 and HSV-2 antigens. Those patients
under 6 months of age responded similarly to each antigen, whereas those who
were older had significantly higher LTRs to HSV-2. Children with CMV infection
that was acquired early had persistently diminished specific LTRs. In contrast,
after neonatal HSV infection, LTRs to HSV were present even in infancy and
became more specific for the infecting type with increasing age. 
PMID: 6271679, UI: 82052026

5b. Zawilinska B et al. [Congenital and acquired cytomegalovirus infection in
infants confirmed by virologic studies]. [Article in Polish] Przegl Lek
1995;52(7):354-7.
          Zakladu Wirusologii, Instytutu Mikrobiologii, Collegium
          Medicum, Uniwersytetu Jagiellonskiego w Krakowie.
ab: Sixty infants in whom clinical symptoms suspected of cytomegalovirus (CMV)
infection were studied. CMV infection was found in 50% of the subjects. The
diagnosis was based on studies of specific antibodies and isolation of the virus
from urine and/or throat swabs. In most of the children the examinations were
repeated several times, and clinical observations continued for 1 to 42 months
(avg. 18 months). IgM-class antibodies were detected in 26 children and in 18
the virus was isolated. In 3 infants, isolation of CMV virus was the only
evidence of active infection. Persisting viruria (avg. 11 months) and long-term
presence of Ig G antibodies, even to 44th month of life were also observed.
Congenital infection was diagnosed in 4 infants; the remaining ones acquired the
infection during the perinatal period or later. In 7 cases transfused blood
cannot be excluded as the source of infection. The clinical symptoms manifested
in infected and non-infected children were similar. There was a statistically
significant increase in the occurrence of hepatomegaly, splenomegaly,
hyperbiliru-binemia and diarrhoea in infected children. Congenital abnormalities
were found in 10 infected children, including 4 cases of congenital cytomegaly. 
PMID: 8525003, UI: 96106106

6. A series of PCR-based studies in the 1990s have demonstrated that
intraneuronal HSV can within the CNS of persons showing no peripheral signs (eg,
persons have anti-HSV antibodies within normal reference range values) and
manifesting no peripheral symptoms such as zoster. A rambling discussion with
many citations can be found at:
          http://www.jorsm.com/~binstock/lks-hsv.htm

7. Fudenberg HH. Dialysable lymphocyte extract (DLyE) in infantile onset autism:
a pilot study. Biotherapy 1996;9(1-3):143-7.
          Neurolmmuno Therapeutics Research Foundation Spartanburg,
          S.C., USA.
ab: 40 infantile autistic patients were studied. They ranged from 6 years to 15
years of age at entry. 22 were cases of classical infantile autism; whereas 18
lacked one or more clinical defects associated with infantile autism
("pseudo-autism"). Of the 22 with classic autism, 21 responded to transfer
factor (TF) treatment by gaining at least 2 points in symptoms severity score
average (SSSA); and 10 became normal in that they were main-streamed in school
and clinical characteristics were fully normalized. Of the 18 remaining, 4
responded to TF, some to other therapies. After cessation of TF therapy, 5 in
the autistic group and 3 of the pseudo-autistic group regressed, but they did
not drop as low as baseline levels.   PMID: 8993773, UI: 97146917

8. One autism-spectrum child's immune panel, as originally presented to the
autism-list of St. Johns University on May 27, 1999:

.....a sample panel from 1997.....
Keep in mind that the following immune panel was one of several categories of
lab data derived on behalf of a particular child. Furthermore, the following
panel presents little about IgE and IgA, which are very important aspects for
many autism-spectrum children, thus this list of tests ought not be considered
"complete and applicable for every autism-spectrum child". 
     I have placed an asterisk by tests whose data have been noteworthy in some
or most of the lab reports I've perused.

* C1Q immune complex  
* Complement C3
* Complement C4
  Conglutinin Solid Phase
  Polyethylene Glycol
  Raji Cell

* HSV1         IgG
* HSV2         IgG
* CMV          IgG
* Varicella    IgG  
* HHV6         IgG

* EBV nuclear antigen antibody
* EBV early diffuse antigen antibody
* EBV Viral Capsid Antigen         IgG
* EBV VCA                          IgM
* EBV early restricted...               

* Candida
* Mumps        IgG
* Measles      IgG (rubeola)
* Rubella      IgG
* Polio 1      IgG
* Polio 2      IgG
* Polio 3      IgG
* Coxsackie viruses A7, A9, A10, A16, B1, B2, B3, B4, B5, B6

* Cortisol RIA in am
* DHEA (gender specific ranges)
* Thyroid-function tests:
     Thyroglobulin antibodies
     Thyroid Peroxidase antibodies
     TSH
     T3 free RIA
     Thyroxine

     [comment: I recommend that parents of autism-spectrum children
     consult with Shirley Adams or Ricci Hedequist regarding competent
     approaches to thyroid-function evaluations and interpretations of
     test results.]

* Interferon alpha -- serum
* Interleukin 1 -- serum

* Further immune-related tests:
     % of CD4+ CD29+               % of CD3+ (T cells)
     # of CD4+ CD29+               # of CD3+ (T cells)
                                   % of CD4+ (T helper cells)
     % of CD4+ CD45RA+             # of CD4+ (T helper cells)
     # of CD4+ CD45RA+             % of CD8+ (T suppressor cells)
                                   # of CD8  (T suppressor cells)
     % of CD4+ CD45RO+        
     # of CD4+ CD45RO+        

* total WBC
* total lymphocytes

* CD4/CD8 ratio, HLA-DR data
     % of CD3+ CD26+               % of CD8+ CD28+
     # of CD3+ CD26+               # of CD8+ CD28+
     % of CD3+ HLA-DR+             % of CD8+ CD38+
     # of CD4+ HLA-DR+             # of CD8+ CD38+          
     % of CD8+ HLA-DR+
     # of CD8+ HLA-DR+

* Natural Killer cell data
     % of CD3- CD16+ CD 56+ (NK cells)
     # of CD3- CD16+ CD 56+ (NK cells)
     NK cell function 

* Lymphocyte stimulation by:
     PHA
     Pokeweed
     Concanavillin

Most of the above assays were performed by Antibody Assay Laboratories (1-800-
522-2611).     I find the AAL lab-test reports to be well presented and contain
summary explanations for most of the immune-tests and atypical findings. In
contrast, some labs present reports that are very minimal in what they present.

The following assays were done by Immunosciences Lab, Inc, whose reports contain
fine delineations for each assay's results. Two doctors ordered tests from this
lab:

Dr. Fudenberg ordered:
     antibodies tests:
          Serotonin antibodies     IgG, IgM
          Dopamine                 IgG, IgM
          Myelin-related MAG       IgM, IgM, IgA
          GM1 ganglioside          
          Anti-sulphatide

A DAN! doc ordered:
     levels of:
*         Interleukin 6
*         Tumor Necrosis Factor alpha
*         Interferon alpha

I have also found the following to be useful, because they provide a baseline
measure of the child's humoral immune function, because occasionally these
levels are mildly or excessively elevated, and/or because some children lack one
of these antibodies despite having been vaccinated:
*    anti-tetanus antibodies
*    anti-diptheria antibodies
*    anti-pertussis antibodies

While the initial number of immune panels analyzed remains small (n<15), their
data suggest: (a) that a thorough immune panel ought be ordered for many autism-
spectrum children, (b) that certain treatment protocols (eg, Prednisone) be
enacted only *after* atypical chronic infections have been ruled out, and (c)
that restoring immune-competence and reducing the level of atypical chronic
infections are legitimate parental and medical goals in these children.