"Threshold-crossing": A Useful Way to Establish the Counterfactual in Clinical Trials?

A central question in the assessment of benefit/harm of new treatments is: how does the average outcome on the new treatment (the factual) compare to the average outcome had patients received no treatment or a different treatment known to be effective (the counterfactual)? Randomized controlled trials (RCTs) are the standard for comparing the factual with the counterfactual. Recent developments necessitate and enable a new way of determining the counterfactual for some new medicines. For select situations, we propose a new framework for evidence generation, which we call "threshold-crossing." This framework leverages the wealth of information that is becoming available from completed RCTs and from real world data sources. Relying on formalized procedures, information gleaned from these data is used to estimate the counterfactual, enabling efficacy assessment of new drugs. We propose future (research) activities to enable "threshold-crossing" for carefully selected products and indications in which RCTs are not feasible.

2012: A watershed year for Alzheimer's disease research.

Despite negative topline phase 3 clinical trial results for bapineuzumab and solanezumab in mild to moderate AD, findings from these trials and recent advances suggest renewed optimism for anti-amyloid therapies. Aß immunotherapy has now demonstrated its ability to engage CNS Aß and modify downstream CNS biomarkers in bapineuzumab treated patients, and to show likely cognitive benefits in mild patients treated with solanezumab. The current availability of potent BACE inhibitors provides additional opportunities to test the value of reducing Aß in the clinic. Trial enhancements, such as selecting and enriching for early stage AD, treating participants longer and using more sensitive composite endpoints may further improve our chances of demonstrating clinical efficacy and securing beneficial treatments for patients.

The effect of nicotine on perceptual, ocular motor, postural, and vegetative functions at rest and in motion.

Nicotine has wellknown, unpleasant side effects, e.g., transient dizziness, nausea, and nicotine-induced nystagmus (NIN). To investigate factors influencing these effects, we addressed three questions: (1) Is the intensity of dizziness, nausea, NIN, and unsteadiness dependent on nicotine dosage? (2) Does the intensity of perceptual, ocular motor, vegetative effects, and postural imbalance correlate? (3) Do visual or vestibular motion stimuli produce and/or aggravate distressing dizziness and nausea? Sixty healthy non-smokers or occasional smokers participated; 40 were tested once before and six times after application of a nicotine nasal spray in doses of 1 mg or 2 mg with or without motion stimulation; 20 received a placebo nasal spray. Plasma nicotine concentrations were significantly related to nicotine dosage. Dizziness, nausea, NIN, and unsteadiness also depended on the nicotine dosage (p < 0.01).Nicotine blood concentration was a better predictor for the temporal dependence of nystagmus than nicotine dosage. Dizziness correlated highly with nausea (R = 0.63, p < 0.001). The degree of nicotine-induced nausea significantly correlated with postural imbalance. The time course of postural sway differed according to nicotine dosage and gender: for women, there was no clear relationship between sway magnitude and nicotine dosage, while men showed increased sway with higher dosage. Motion stimulation increased nicotine-induced dizziness and nausea, but did not significantly influence NIN or postural imbalance. Our data support the view that all measured adverse effects reflect dose-dependent nicotine-induced vestibular dysfunction. Additional motion stimulation aggravates dizziness and nausea, i.e., nicotine increases sensitivity to motion sickness.

The association of post-stroke neurological improvement with risk of subsequent deterioration due to stroke events.

We sought to simultaneously confirm that substantial recovery at day 1 and day 7 after acute ischaemic stroke onset is associated with subsequent neurological deterioration in patients of the Acute Stroke Therapy by Inhibition of Neutrophils randomized clinical trial. Substantial recovery was assessed by improvement in the National Institutes of Health Stroke Score (NIHSS). Neurological deterioration was defined as any stroke event or NIHSS worsening from recovery assessment to day 90. After adjusting for age, t-PA and day 1 NIHSS, there was a non-significant tendency of substantial (pre-specified as 75%) recovery at day 1 to be associated with later deterioration [odds ratio (OR) 2.47; 95% CI, 0.95-6.50]. The corresponding OR for substantial (pre-defined as 65%) recovery at day 7 was 1.84 (0.85-3.96). Other thresholds for recovery were significantly associated with later deterioration: >50%, 80%, 90% and 100% for day 1 and >50%, 60%, 70%, 90% and 100% for day 7. The effect of recovery at day 1 was more important than that of later recovery. This study confirms the association between recovery and subsequent neurological deterioration and is the first to indicate the greater importance of acute recovery at day 1 in comparison with later recovery.

[Proliferation and hTERT expression in neuroblastoma]. / Proliferationskinetik und hTERT-Expression beim NeuroblastomUntersuchungen zum Zusammenhang.

BACKGROUND: Transcription of the catalytic subunit of telomerase, human Telomerase Reverse Transcriptase (hTERT), and increased tumor cell proliferation are powerful prognostic factors in neuroblastoma. We therefore investigated their relationship in a large group of neuroblastomas. METHODS: RT-PCR analysis was used to discriminate between the various hTERT transcripts. Tumor cell proliferation was assessed immunohistochemically using two different cell-cycle specific antibodies and the results were compared by statistical analysis. RESULTS AND CONCLUSIONS: 54 out of 115 neuroblastomas showed hTERT transcripts, 25 of which also possessed full-length transcripts. Full-length hTERT transcripts were correlated with MYCN-amplification, with a Ki67-proliferation index > or = 25% and a repp86-proliferation index > or = 10% (p<0,0001), but only a Ki67-proliferation index > or = 25% was associated with general hTERT transcription (p=0,001). Our data confirm the close relationship between hTERT transcription and tumor cell proliferation and further strengthen the exceptional prognostic power of the repp86-proliferation index.

Telomerase activity in B-cell non-Hodgkin lymphomas is regulated by hTERT transcription and correlated with telomere-binding protein expression but uncoupled from proliferation.

Telomere maintenance is a prerequisite for immortalisation, and in most malignant cells is carried out by telomerase, an enzyme that synthesis new telomeric repeats on the chromosome ends. In normal or reactive tissues with a high regenerative capacity, telomerase is regulated according to the telomere loss that occurs during proliferation. To evaluate the interaction of proliferation and telomerase activity in malignant lymphomas, we quantified telomerase expression in different non-Hodgkin lymphomas in comparison to normal or reactive lymph nodes. Surprisingly, the activity levels were the same in most of the lymphomas analysed as compared to reactive lymph nodes. Significantly higher activity was detected only in Burkitt's lymphoma. Telomerase activity correlated well with hTERT and c-myc expression, but was independent of proliferation. To evaluate interactions of telomere-binding protein expression on telomerase expression in non-Hodgkin lymphoma, the mRNA levels of TRF1, TRF2, tankyrase and hPif1 were assessed by real-time RT-PCR. We demonstrate here that the magnitude of telomerase upregulation does not necessarily reflect the requirement of telomere compensation caused by proliferation. Telomerase regulation in non-Hodgkin lymphomas is therefore uncoupled from proliferative stimuli found in reactive lymphoid tissue. We suggest that the upregulation of specific telomere-binding proteins like TRF2 may contribute to telomere maintenance in malignant lymphoma.

Regulation of telomerase activity by alternate splicing of human telomerase reverse transcriptase mRNA in a subset of neuroblastomas.

It has been proposed that the regulation of telomerase takes place at the transcriptional level, the expression of the catalytic subunit human telomerase reverse transcriptase (hTERT) being crucial for telomerase activity (TA). Recently, differential splicing of hTERT mRNA has been demonstrated in various tissues during embryonal development, and it has been suggested that only full-length transcripts translate into functionally active telomerase. With this in view, we analyzed the different hTERT transcripts by reverse transcriptase-polymerase chain reaction in neuroblastic tumors and compared the results with the TA, the tumor growth fraction, and the MYCN status. In a series of 38 neuroblastic tumors, high TA and full-length hTERT transcripts were found in nine samples, whereas nine samples showed absence of both enzymatic activity and hTERT transcripts. Interestingly, in another eight samples, low or absent TA coincided with a lack of full-length hTERT transcripts. Eleven samples contained hTERT transcripts with low or undetectable TA and one sample had low TA but no hTERT transcripts. TA correlated with MYCN amplification and was weakly associated with the proliferative activity. Moreover, a significant correlation with tumor progression was observed. Our findings point at a posttranscriptional regulation of TA in a subset of neuroblastic tumors. Because high TA was detected only in tumors with full-length hTERT transcripts, reverse transcriptase-polymerase chain reaction analysis of archival neuroblastic tumor samples might help to appraise the malignant potential in individual cases.

The attentional role of the left parietal cortex: the distinct lateralization and localization of motor attention in the human brain.

It is widely agreed that visuospatial orienting attention depends on a network of frontal and parietal areas in the right hemisphere. It is thought that the visuospatial orienting role of the right parietal lobe is related to its role in the production of overt eye movements. The experiments reported here test the possibility that other parietal regions may be important for directing attention in relation to response modalities other than eye movement. Specifically, we used positron emission tomography (PET) to test the hypothesis that a 'left' parietal area, the supramarginal gyrus, is important for attention in relation to limb movements (Rushworth et al., 1997; Rushworth, Ellison, & Walsh, in press). We have referred to this process as 'motor attention' to distinguish it from orienting attention. In one condition subjects spent most of the scanning period covertly attending to 'left' hand movements that they were about to make. Activity in this first condition was compared with a second condition with identical stimuli and movement responses but lacking motor attention periods. Comparison of the conditions revealed that motor attention-related activity was almost exclusively restricted to the 'left' hemisphere despite the fact that subjects only ever made ipsilateral, left-hand responses. Left parietal activity was prominent in this comparison, within the parietal lobe the critical region for motor attention was the supramarginal gyrus and the adjacent anterior intraparietal sulcus (AIP), a region anterior to the posterior parietal cortex identified with orienting attention. In a second part of the experiment we compared a condition in which subjects covertly rehearsed verbal responses with a condition in which they made verbal responses immediately without rehearsal. A comparison of the two conditions revealed verbal rehearsal-related activity in several anterior left hemisphere areas including Broca's area. The lack of verbal rehearsal-related activity in the left supra-marginal gyrus confirms that this area plays a direct role in motor attention that cannot be attributed to any strategy of verbal mediation. The results also provide evidence concerning the importance of ventral premotor (PMv) and Broca's area in motor attention and language processes.

Cerebral dominance for action in the human brain: the selection of actions.

PET was used to study cerebral dominance for the selection of action. In one condition the subjects moved one of two fingers depending on the cue presented (choice reaction time), and in another they moved the same finger whatever the cue (simple reaction time). There was also a baseline condition in which cues were shown but no movements were made. A conjunction analysis was performed to reveal those areas which were more activated for the choice versus simple reaction time, irrespective of whether the right or left hand was used. The activations were in prefrontal, premotor and intraparietal areas, and they were all in the left hemisphere. Thus, while there were activations in the right hemisphere for the choice versus simple reaction time task when the subjects used their left (contralateral) hand, there were activations in left prefrontal, premotor and parietal areas whether the right (contralateral) or left (ipsilateral) hands were used. It is argued that the results suggest that the left hemisphere is dominant not only for speech but also for action in general.

In situ reverse-transcriptase polymerase chain reaction demonstration of the EWS/FLI-1 fusion transcript in Ewing's sarcomas and peripheral primitive neuroectodermal tumors.

It is now widely accepted that the EWS/FLI-1 fusion transcript is associated with tumors of the Ewing family. To test whether it is possible to detect the fusion transcript by means of combining polymerase chain reaction (PCR) methodology and immunohistochemistry, we investigated tumors of the Ewing family using in situ reverse transcriptase (RT)-PCR. We were able to demonstrate the t(11;22) fusion transcript in five of six cases of Ewing's sarcoma and four of four peripheral primitive neuroectodermal tumors. These results were confirmed using fluorescence in situ hybridization in seven tumor samples. In situ RT-PCR-labeled fusion transcripts were found in virtually all tumor cells within a given sample, indicating that each cell possessed the t(11;22) transcript. We conclude from these results that in situ RT-PCR can be used for the rapid detection of EWS/FLI-1 fusion transcripts in biopsy material. The findings also suggest that all cells of the tumors of the Ewing family carry the EWS/FLI-1 fusion transcript.

Absence of 9q22-9qter in trisomy 9 does not prevent a Dandy-Walker phenotype.

We report on a female fetus with partial trisomy 9 due to a reciprocal translocation in the mother. Routine ultrasound examination at 23 weeks showed hypoplasia of the cerebellar vermis, dilated foramen Magendii, and dilatation of the cisterna magna. Due to the poor prognosis, the parents opted for termination of pregnancy. A postmortem examination confirmed caudal hypoplasia and dysplasia of the cerebellar vermis, resulting in a massively dilated foramen Magendii through which the enlarged cisterna magna communicated with the fourth ventricle. There was also micropolygyria indicating migration disorder. Cytogenetic studies showed a 47,XX,+der(9)t(7;9) (q35;q22.2)mat karyotype. Investigation of the parents revealed a translocation (7;9) (q35;q22.2) in the mother and a normal male karyotype in the father. We systematically searched the chromosome 9 gene map for genes that were trisomic in our fetus and genes that were located on the regions that had the normal two copies of genes. Genes that could potentially be involved in the formation of the Dandy-Walker phenotype are transcription factors or genes responsible for the regulation of normal in particular cerebral development but also adhesion molecules. We conclude that one cause for Dandy-Walker malformation could be a gene dosage effect of genes located on 9pter-9q22. In addition, it seems that absence of trisomy 9 in q22-pter does not prevent abnormal cerebellar development.

The neuroanatomy of autism: a voxel-based whole brain analysis of structural scans.

Autism is a biological disorder which affects social cognition, and understanding brain abnormalities of the former will elucidate the brain basis of the latter. We report structural MRI data on 15 high-functioning individuals with autistic disorder. A voxel-based whole brain analysis identified grey matter differences in an amygdala centered system relative to 15 age- and IQ-matched controls. Decreases of grey matter were found in anterior parts of this system (right paracingulate sulcus, left inferior frontal gyrus). Increases were found in posterior parts (amygdala/peri-amygdaloid cortex, middle temporal gyrus, inferior temporal gyrus), and in regions of the cerebellum. These structures are implicated in social cognition by animal, imaging and histopathological studies. This study therefore provides converging evidence of the physiological basis of social cognition.

A functional anatomy of anticipatory anxiety.

Anticipatory anxiety is a complex combination of a future-oriented cognitive state, negative affect, and autonomic arousal. A dual-task paradigm of anticipation of electric shocks and a motor-learning task was used to examine the changes in neural patterns of activation associated with modulation of the cognitive state in anxiety by a distracting motor task. We used positron emission tomography (PET) and 15O-water to measure regional cerebral blood flow (rcbf) in 10 healthy male volunteers. A 2x2 factorial design-(shock vs no shock) x (low vs high distraction) was used with three scans per condition. Twelve PET scans were performed on each subject. In six of these scans, subjects were given electric shocks. In all scans, subjects also simultaneously performed a motor repetition (low distraction) or learning (high distraction) task. Galvanic skin conductance (GSR), Spielberger State and Trait Anxiety Inventory (STAI), and self-report data were also collected. In comparisons between the shock and no-shock conditions, the main finding was of increased rcbf in the left insula (-38,8,8) (z = 4.85, P<0.05 corrected) and a homologous area in the right insula at a lower threshold (z =3.20, P = 0.001 uncorrected). Other areas activated were the right superior temporal sulcus, left fusiform, and left anterior cingulate. Using the STAI-state scores as a covariate of interest, significant correlations with rCBF were seen in the left orbitofrontal cortex, left insula, and left anterior cingulate cortex. There was no significant distraction effect as measured by the STAI, self-report, GSR response or interactional analysis of the PET data. These findings support the role of paralimbic structures as neural substrates of anticipatory anxiety. The failure to demonstrate behavioral and neurophysiological changes with the distractor task may reflect the modest increases in anxiety with the shock, the relatively simple distractor task, and small sample size.

Kallmann's syndrome: mirror movements associated with bilateral corticospinal tract hypertrophy.

OBJECTIVE: To investigate the etiology of mirror movements in patients with X-linked Kallmann's syndrome (xKS) through statistical analysis of pooled white matter data from structural MR images. BACKGROUND: Mirror movements occur in 85% of xKS patients. Previous electrophysiologic studies have suggested an abnormal ipsilateral corticospinal tract projection in xKS patients exhibiting mirror movements. However, an alternative hypothesis has proposed a functional lack of transcallosal inhibitory fibers. METHODS: T1-weighted brain scans were normalized into stereotaxic space with segregation of gray and white matter to allow comparison of pooled white matter data on a voxel-by-voxel basis using SPM-96 software. Nine xKS patients were compared with two age-matched groups of nonmirroring individuals: nine patients with autosomal Kallmann's syndrome (aKS) and nine age-matched normal (healthy) men. RESULTS: Hypertrophy of the corpus callosum was found in both Kallmann's syndrome groups: the anterior and midsection in xKS, and the genu and posterior section in aKS. Bilateral hypertrophy of the corticospinal tract was found only in the group of xKS patients exhibiting mirror movements. SPM analysis was validated by an independent region of interest analysis of corpus callosum size. CONCLUSION: Although morphometry on its own cannot determine the cause of mirror movements, the specific finding of a hypertrophied corticospinal tract in xKS is consistent with electrophysiologic evidence suggesting that mirror movements in xKS result from abnormal development of the ipsilateral corticospinal tract fibers.

The time course of changes during motor sequence learning: a whole-brain fMRI study.

There is a discrepancy between the results of imaging studies in which subjects learn motor sequences. Some experiments have shown decreases in the activation of some areas as learning increased, whereas others have reported learning-related increases as learning progressed. We have exploited fMRI to measure changes in blood oxygen leve-dependent (BOLD) signal throughout the course of learning. T2*-weighted echo-planar images were acquired over the whole brain for 40 min while the subjects learned a sequence eight moves long by trial and error. The movements were visually paced every 3.2 s and visual feedback was provided to the subjects. A baseline period followed each activation period. The effect due to the experimental conditions was modeled using a square-wave function, time locked to their occurrence. Changes over time in the difference between activation and baseline signal were modeled using a set of polynomial basis functions. This allowed us to take into account linear as well as nonlinear changes over time. Low-frequency changes over time common to both activation and baseline conditions (and thus not learning related) were modeled and removed. Linear and nonlinear changes of BOLD signal over time were found in prefrontal, premotor, and parietal cortex and in neostriatal and cerebellar areas. Single-unit recordings in nonhuman primates during the learning of motor tasks have clearly shown increased activity early in learning, followed by a decrease as learning progressed. Both phenomena can be observed at the population level in the present study.

The preparation, execution and suppression of copied movements in the human brain.

We used positron emission tomography (PET) to measure movement set-related changes in regional cerebral blood flow (rCBF) when human subjects were asked to copy hand movements. Movement set-related activity in the brain is thought to reflect the processes of movement selection, preparation and inhibition. Four conditions were used. In the first condition, prepare and execute (PE), the hand stimulus to be copied was shown to subjects 3 s before an auditory "go"-cue instructed subjects to execute the movement; a large part of the scanning time was therefore spent in preparing to move. In the immediate execution condition (E), the hand stimulus and the go cue were presented simultaneously. The prepare-only condition (P) was similar to PE, except subjects only prepared to make the movement and did not actually execute any movement when they heard the auditory go-cue. The same stimuli were presented in a baseline condition (B), but the subjects were instructed to neither prepare nor execute movements. There were 5 principle findings: (1) In contrast to a previous study of human set-related activity in which movements were instructed by an arbitrary pattern of LEDs, preparing to make a copied movement causes rCBF changes in area 44 in posterior Broca's area; (2) set-related activity can be recorded in the cerebellar hemispheres and midline; (3) we confirmed that the supramarginal gyrus has a general role in preparing movements - there was more rCBF in the P than the E condition; (4) the cerebellar nuclei and the basal ganglia may be particularly involved in the initiation and execution of a planned movement; these regions were more active in the PE condition than the P condition; (5) the ventrolateral prefrontal cortex and a left anterior cingulate area are part of a distributed system involved in the suppression of a motor response; these areas were significantly more active in the P than the PE condition.

[Rhabdomyosarcoma and extraosseous Ewing's sarcoma]. / Rhabdomyosarkome und extraossäre Ewing-Sarkome.

Rhabdomyosarcomas (RMS) and extraosseous Ewing's sarcomas (EOE) including malignant peripheral neuroectodermal tumors (MPNT) are the most frequent soft tissue malignancies of childhood. They account for 60.2% of 2.350 cases collected in the files of the Kiel Pediatric Tumor Registry. RMS: It is absolutely necessary to distinguish between embryonal (e) and alveolar (a) RMS, since these are two distinct tumor entities with significant differences in clinical presentation, morphology, molecular biology, cytogenetics and prognosis (Botryoid and spindle cell RMS are special variants of eRMS). The overall proportion of eRMS: aRMS is 2.4:1. Most cases of eRMS develop in the first 10 years of life (77.3%) while the age distribution of aRMS is almost constant in childhood and adolescence. Embryonal RMS exhibit a significantly higher proportion of male patients than aRMS (m:f = 1.72:1 vs. 1.06:1). A higher percentage of aRMS cases (25%) shows metastatic disease at the time of diagnosis than eRMS (8%), and the overall survival rates of aRMS are significantly lower even in localised disease (stage I-III) than in eRMS (59% vs. 76%; p < 0.002) (Data from the Cooperative Soft Tissue Sarcoma Study CWS). EOE: Despite proven histogenetic relationship (identical chromosomal rearrangements and fusion genes) the members of the Ewing's sarcoma (ES) family, classic ES and MPNT, whether osseous or extraosseous display significant differences in location, morphology and prognosis. Morphologically, MPNT and classic ES can be considered to be the extremes of a spectrum with overt neurodifferentiation in the former and lack of neural differentiation in the latter. Matched-pairs analysis of CWS EOE and MPNT cases show dramatically more unfavorable overall survival rates in MPNT than in EOE (45% vs. 67%).

Mirror movements in X-linked Kallmann's syndrome. I. A neurophysiological study.

Possible mechanisms underlying the pathological mirror movements that are seen in the majority of patients with X-linked Kallmann's syndrome have been investigated using neurophysiological techniques. An EMG was recorded from the first dorsal interosseous muscle (1DI) during voluntary self-paced abduction of one indexed finger; EMG activity could also be recorded simultaneously from the contralateral 1DI. There was no significant difference between the time of onset of the bursts of voluntary and involuntary mirroring EMG. Focal magnetic stimulation of the hand area of the motor cortex revealed the presence of fast conducting bilateral corticospinal projections from each motor cortex in all subjects. However, both inter- and intra-subject differences exist when considering the ratio of ipsilaterally to contralaterally projecting axons. Cross-correlation analysis of multi-unit EMGs recorded during simultaneous voluntary sustained activation of homologous left and right pairs of distal upper limb muscles was performed. A short duration central peak was seen in the cross-correlograms indicating the presence of a common drive to left and right homologous motor neuron pools. This common drive may result from the synchronous activation of intermingled ipsilaterally and contralaterally projecting corticospinal neurons in the motor cortex. Cutaneomuscular reflexes were recorded from the 1DI following stimulation of the digital nerves of the index finger. Typically each reflex comprises spinal and longer latency trans-cortical components. In these subjects, the long latency components of the reflex response could, in addition, be recorded from the 1DI of the non-stimulated side. We conclude that these subject have a novel ipsilateral at least in part, for the pathological mirroring.

Mirror movements in X-linked Kallmann's syndrome. II. A PET study.

To investigate the mechanism of mirror movements seen in X-linked Kallmann's syndrome, we measured changes of regional cerebral blood flow with H2 15O-PET. We studied six right-handed Kallmann male subjects and six matched, right-handed control subjects during an externally paced finger opposition task. The analyses were done both on a single subject and a group basis. The Kallmann group showed a strong primary motor cortex (M1) activation contralateral to the voluntarily moved hand, but there was also a significant degree of M1 activation ipsilateral to the voluntarily moved hand, i.e. contralateral to the mirroring hand. However, when comparing contralateral to ipsilateral M1 activation, the M1 activation contralateral to the voluntarily moved hand was significantly stronger. In the controls, significant increases in rCBF were seen in the contralateral M1 during voluntary movement of either hand; a small ipsilateral M1 activation was found in two out of six normal subjects when they moved their left hand. In a second experiment it was shown that, in two out of two Kallmann subjects, passive movements of the right hand resulted in left M1 activation that was similar to the activation in the left M1 when subjects made mirror movements with their right hand. This suggests, but does not prove, that the small but significant activation of the ipsilateral M1 in Kallmann's subjects may be due to sensory feedback from the involuntarily mirroring hand.

Detection of rare RNA sequences by single-enzyme in situ reverse transcription-polymerase chain reaction. High-resolution analyses of interleukin-6 mRNA in paraffin sections of lymph nodes.

To study the distribution pattern of interleukin-6 (IL-6)-producing cells in normal human lymph nodes, we applied the in situ reverse transcription-polymerase chain reaction technique. We describe a new modification of this technique for monitoring small amounts of specific nucleotide sequences in conventional paraffin sections. This technique differs in at least two respects from those described earlier. The two decisive steps are: 1) the reverse transcription of mRNA and the subsequent amplification of cDNA by polymerase chain reaction are performed by a new single enzyme capable of both reaction types in one and the same medium without buffer exchange; and 2) for the specific detection of the amplified cDNA, a modified version of the primed in situ labeling technique was used. The technique, carried out on normal human lymph nodes, traces a low load of IL-6 mRNA in fibroblasts, endothelial cells, and a minor population of T lymphocytes in the pulp region. High levels of expression were encountered in about 20% of perisinusoidal pulp macrophages. In addition, moderate activity was detectable in sinus lining cells. Because no major activity was found in the germinal centers of the lymphoid B follicles and in the T zone, it is suggested that the plasma cell differentiation ensuing from primary and secondary B-cell immunization is mainly effected by the sinus lining cells as well as perifollicular and perisinusoidal pulp macrophages capable of producing high amounts of IL-6.