742 Proc. roy. Soc. Med. Volume 66 August 1973
`
`(3) Diminished fibrinolytic properties of the blood
`which would promote fibrin deposition and
`enhance fibrin deposition and clotting.
`
`This tendency to abnormal stasis and clotting
`of blood in the superficial dermal venules in
`acrocyanosis (Crocq 1896) and the resulting
`alteration in the anatomy of the blood vessels in
`the upper dermis can explain the associated signs
`of micropapular chilblains (erythema pernio
`papulosum, Haxthausen 1930, Hutchinson 1878),
`pink cinnabar spots (Stem 1937), and reticular
`patterning (Estes 1956). Micropapular chilblains
`are visible on the skin when the clotting has
`affected a sufficiently large cluster of microvessels.
`
`Since Sir Thomas Lewis (1927), many have
`tried to explain the signs of acrocyanosis on the
`basis of: functional disorder of some segment of
`acral blood vessels whether arterial, capillary or
`venous; pharmacological oddity of the cutaneous
`nerves, their endings or responses; a perversion
`of the action of vasoactive and other agents in the
`skin; occult immunological irregularities; and
`other physiological and pathological peculiarities.
`Burch & Phillips (1963), exhaustively reviewing
`all these writings, concluded that the signs could
`not be satisfactorily explained on such grounds
`alone.
`
`I postulate that the signs in the skin of acro-
`cyanosis or micropapular chilblains are merely
`different degrees of the same disorder. They
`result from an accentuation of the normal features
`of healthy whole blood flow, mainly stasis and
`clotting on exposure to cold. In these conditions
`the hyperviscosity of the blood allows clotting to
`occur only in the cutaneous microvessels exposed
`to the cold.
`
`The disease, then, results from an intravascular
`defect rather than a blood vessel or skin disorder
`in the first instance. This blood coagulation
`disorder is probably not a simple matter but the
`result of a complex and interrelated series of
`defects. It only involves the skin and affects no
`other organs because they are at body tempera-
`ture. If it were not for the special circumstances
`of its exposure to surface chilling in the skin
`microvessels, the blood's defects would have
`been sufficiently minor to have escaped detection.
`REFERENCES
`Burch G F & Phillips J {1963) In: Handbook ofPhysiology:
`Section 2. Ed. W F Hamilton. American Physiological
`Society, Washington; 1,1230-32
`CopemanPWM
`{1970) British Journal of Dermatology 81, Suppl. 5, 5 I
`Copeman P WM & Ryan T J
`(1971) British Journal of Dermatology 85, 205
`Croeq C {1896) Semalne midicale (Paris) lei, 298
`Estes J (19S6) Motkrn Concepts of Cardiovascular Disease 15, 355
`Haxtbauaen H (1930) Cold in Relation to
`Skin Diaeaaes. Heinemann, London; p 6S
`
`36
`
`Hutchinson J ( 1878) Lectures on Clinical Surgery Vol. I:
`On Certain Rare Diseases of the Skin. Churchill, London; p 362
`Lewis T H (1927) The Blood Vessels of the Human Skin and
`Their Responses. Shaw & Son, London
`Ryan T J & Copeman P W M
`(1969) British Journal of Dermatology 81, S63-73
`Stem S {1937) British Journal of Dermatology 49, 100-106
`Zweifach B W & Metz DB (19SS) Angiology 6, 281-290
`
`Histogenesis of the Inflammatory
`Component of Rosacea
`by R Marks sse MRCP
`(Institute of Dermatology,
`StJohn's Hospital for Diseases of the Skin,
`Lisle Street, Leicester Square, London WC2H 7 BJ)
`
`Rosacea is characterized by a wide range of
`physical signs. On the one hand is erythema and
`telangiectasia making up the vascular component,
`and on the other papules and pustules which
`constitute the inflammatory aspect of the disease.
`It is impossible to say which of these components
`is primary or even whether the inflammation and
`the vascular components are causally related.
`
`In a previous investigation it was shown that
`the hair follicles are not the initial focal points of
`the inflammation so characteristic . of papular
`rosacea(Marks & Harcourt-Webster 1969). When
`there is infiltration and disruption or even abscess
`formation in the follicular epithelium it seems to
`be a secondary process and not of fundamental
`importance in the overall biology of the disorder.
`As the inflammation does not appear to be the
`result of a follicular disorder,
`the question
`remains - what is the cause of the inflammation?
`
`Detailed examination of biopsy material from
`patients with papular rosacea has shown the
`cellular infiltrate to be predominantly distributed
`around the small blood vessels of the subpapillary
`venous plexus (Marks & Harcourt-Webster 1969).
`In addition, in disseminated rosacea there is
`pronounced vascular involvement amounting to
`a frank vasculitis in places (Marks & Wilson
`Jones 1969). Furthermore, telangiectasia and the
`cedema that is so commonly observed add to the
`suspicions that the blood vessels are more than
`incidentally affected and primary vascular involve-
`ment could be central to the pathogenesis of the
`inflammation. For these reasons, the role of small
`blood vessels in papular rosacea has been investi-
`gated in this study by histochemical and auto-
`radiographic techniques. At the same time, and
`as a corollary to the blood-vessel studies, the rate
`of proliferation and the enzyme histochemical
`characteristics of the cellular infiltrate have been
`examined.
`
`Dr. Reddy's Laboratories, Ltd., et al.
`v.
`Galderma Laboratories, Inc.
`IPR2015-__
`Exhibit 1035
`
`Exh. 1035
`
`

`
`37
`
`Secti011 of Dermato/ogy
`
`743
`
`Patientsand Methods
`Altogether 27 patients were studied. All had
`typical papular rosacea with a background of
`erythema and
`telangiectasia and a variable
`amount of swelling. There were 15 men and 12
`women with an average age of 52.6 years (range
`24-77). Seven patients were taking tetracycline at
`the time of biopsy; 2 had started two days pre-
`viously and the other 5 had been treated for
`periods of one to three weeks. Seven other
`patients had used topical corticosteroids for vary-
`ing periods. Biopsies were taken of papules with
`a5mmpunch.
`
`In order to identify cells about to divide by
`autoradiography, 14 papules were injected intra-
`cutaneously with tritiated thymidine. Ten papules
`were removed one hour after injection. The other
`four biopsies were removed at intervals of one,
`two, three and four days after injection. Ilford
`nuclear emulsion (K4) was used to coat the
`sections, and exposure times of two and three
`weeks were employed.
`
`To give more precision to the kinetic data of
`the cells the inflammatory infiltrate was divided
`into three main cellular types : lymphocytes,
`histiocytes and endothelial cells. The number of
`cells Iabelied in each category was counted
`(between 500 and 1000 cells of each type for every
`specimen) and expressedas a percentage (labelling
`index). Cells difficult to classify were not counted.
`
`Enzyme histochemical tests on the biopsies of
`16 patients with papular rosacea were performed
`as follows: (1) to test enzymes concerned with
`respiratory activity - NADH diaphorase, lactic
`dehydrogenase (LDH) glucose-6-phosphate de-
`hydrogenase (using the methods in Pearse 1960);
`(2) to test the integrity of the small vessel endo-
`thelium - alkaHne phosphatase (Gomori 1952),
`adenosine triphosphatase (W achstein & Meisel
`1960).
`
`Resu/ts and Comment
`Table 1 shows the mean labeHing indices for the
`three individual cell types in the ten specimens
`removed one hour after injection with tritiated
`thymidine. The overalllabelling index of 3.3% is
`high and suggests a rapid cell turnover rate (Fig
`
`Fig 1 Autoradiograph/rom biopsy of lesion ofpapu/ar
`rosacea injected one hour previously with tritiated
`thymidine. There are many Iabelied inf/ammatory
`cel/s (indicated by arrows). H&E. X 100
`
`1). Lachapelle (1972), using an in vitro method,
`found that the infiltrate in primary irritant derma-
`titis had a labelling index of 0.5% compared with
`2.5% in allergic contact dermatitis. Furthermore,
`the figure of 3.3% obtained in the present investi-
`gation is higher than the range of values given by
`Spector (1971) as being characteristic of a 'low
`turnover' granuloma (0.5-2.0 %) but below the
`value given by that author as an example of a
`high turnover granuloma(4.6%). Thehigh overall
`labelling index is presumably the result of a rapid
`rate of cell destruction but does not imply any
`particular mechanism for cell death. Similar pro-
`liferative responses have been noted by Monis
`et al. (1968) in carrageenan granulomata and by
`Epstein & Krasnabrod (1968) in silica granulo-
`mata. Any actively phagocytic infiltrate may have
`a labelling index of the same order as that
`observed in the present investigation.
`
`Table 2 sets out the values obtained for the
`labelling indices in the four biopsies removed
`between one and four days after intracutaneous
`injection with tritiated thymidine. It shows that
`there was a tendency for the total number of
`Iabelied cells to drop with increasing intervals
`from the time of injection. This observationalso
`
`Table 1
`Mean labelling indices for three cell types in ten specimens
`removed one hour after injection with tritiated thymidine
`
`Table2
`Values for labelling indices in four biopsies removed between one and
`four days alter intracutaneous injection with tritiated thymidine
`
`Gelltype
`Lymphocytes
`Histiocytes
`Endothelial cells
`Alleeiltypes
`
`Mean lahelling
`Index(%)
`3.1
`4.6
`1.3
`3.3
`
`Standard e"or
`o/mean
`0.6
`0.9
`0.3
`o.s
`
`Label/ing indicesfor cell types (%)
`Interval Lymphos. Histios. Endos. All
`1 day
`3.1
`2.8
`3.1
`3.0
`2days
`0.6
`0.3
`5,6
`2.1
`3days
`0.4
`2.4
`2.0
`1.0
`4days
`1.2
`3.1
`2.0
`1.8
`
`Exh. 1035
`
`

`
`744 Proc. roy. Soc. Med. Volume 66 August 1973
`
`38
`
`perithelial cells showed an appreciable number of
`dividing cells suggests that these vessels, though
`'irritated', were not severely damaged and retained
`the capacity for division. The small blood vessels
`were weil delineated with the adenosine triphos-
`phatase and alkaHne phosphatase enzyme histo-
`chemical tests and the mitochondrial enzyme
`reactions. This retention of enzYme reactivity also
`suggests that the small vessels were not signifi-
`cantly injured.
`From the foregoing it seems that the popula-
`tion kinetics and the histochemical characteristics
`of the infiltrate both suggest an actively phago-
`cytic role for these cells. The observations also
`imply intimate vascular participation yet absence
`of a vasculitis. This interpretation of the.data aids
`in understanding the pathogenesis of the in-
`ßammatory reaction in · rosacea ü some such
`mechanism as was suggested by HaJC.thausen
`(1930) . is invoked. He suggested that small
`cutaneous blood vessels were in some way
`damaged by exposure to ßuctuations of environ-
`mental temperature and other climatic stimuli.
`More elastotic change is found in the biopsies of
`rosacea patients than in age, sex and site-matched
`control biopsies (Marks & Rarcourt-Webster
`1969). Small vessels become dilated in elastotic
`tissue. lf these vessels were in addition in some
`way injured, for example by cold, they would also
`become leaky. In rosacea it is possible that as
`
`Fig 3 Autoradiograph showing a /arge di/ated
`thin-walled blood vesse/ with two Iabeiied
`endothelia/ ce//s (indicated by arrows).
`lf.4c-e .. >Slß5
`
`Fig 2 Autoradiograph/rom papular rosacea to show
`Iabelied cells of the histiocytic series. H & E. X 185
`implies a high rate of cell destruction in the
`infiltrate. Larger histiocytic types were Iabelied to
`a greater extent than lymphocytes in several
`specimens (Fig 2).
`Spector & ·Lykke (1966) have shown that
`in experimental granulomatous infiam.mation
`apparently well-differentiated macrophage types
`remained capable of division. All the biopsies
`examined contained !arger histiocytic types that
`reacted quite strongly in the nonspecific esterase
`reaction and were also strongly reactive for the
`NADH and LDH enzyme reactions. These posi-
`tive reactions are characteristic of functional
`phagocytic cells.
`There was a surprising degree of labelling of
`endothelial cells in the autoradiographs prepared
`from biopsy specimens injected one hour pre-
`viously with tritiated thymidine. It is difficult to
`identify endothelial cells in routine histological
`sections. However, by restricting the scoring of
`endothelial cells to spindle-shaped cells lining
`vascular spaces this diffi.culty has been at least
`partially overcome. It was found that endothelial
`cells identified in this way bad a labelling index of
`1.3% (Fig 3). There have been very few studies of
`the proliferation kinetics of vascular endothelium
`and · even ·fewer of · capillary endotheliuni in
`inflamed human skin, so that the significance of
`this finding is uncertain. Not only were endo-
`thelial cells labelled but labelled perithelial cells
`were also obvious in many of the 'one hour'
`~utora~~~~_ap!!~.· · J'f.le .f~c~ ·-t~~t -~!J.dothelial and
`
`Exh. 1035
`
`

`
`39
`
`a consequence of vessel malfunction macro-
`molecules leak into the perivascular tissues and
`attract phagocytes. Furthermore, if fibrinogen
`was amongst these macromolecules, episodes of
`inflammation would result and the pathogenesis
`of the inflammation in rosacea be explained.
`
`REFERENCES
`Epatein W L & Krasnabrod H
`(1968) Lahoratory lnvestigation 18, 190
`Gomori I (1952) International Review ofCytology 1, 323
`Haxthauaen H (1930) BritishJournal o/ Dermatology 42, 105
`LacbapeUe J M (1972) British Journal of Dermatology 87, 460
`MarlaJ R & Harcourt· WebsterN
`(1969) Archives of Dermatology 100, 683
`Marks R & Wilson Jones E
`(1969) Brltish Journal of Dermatology 81, 16
`Monis B, WeinbergT & Spector G J
`(1968) Brltish Journal of Experimental Pathology 49, 302
`Pearse AG E (1960) Histochcmistry: Thcorctical and
`Applied. Churchill, London
`SpectorWG
`(1971) Proceedings ofthe Royal Society of Medicine 64,941
`Spector W G & Lykke A W J
`(1966) Journal o/Pathology and Bacterlology 92, 163
`Wachstein M & Meisel E
`(1960) Journal of Hlstochemistry and Cytochemistry 8, 387
`
`Chronic Fungal Infection
`(E. jloccosum), Erythroderma,
`Immune Deficiency and Lymphoma
`by GM Levene MB MRCP (St John's Hospitalfor
`Diseases ofthe Skin, London WC2H 7BJ)
`
`This report concerns a man with multiple patho-
`logy. An attempt will be made to link bis dis·
`orders together, whicb seem unlikely to have
`arisen solely by unhappy coincidence.
`He is a civil engineer aged 36. He had a healtby
`childhood and the only early comment of note is
`tbat be had some scaling between his toes whilst
`at school. His main trouble started in 1957 whilst
`be was serving in the army in Cyprus at tbe age of
`20. He developed a boil ori his shin, followed by
`pustules on tbe feet and then a generalized weep-
`ing dermatitis. He was treated first in Nicosia and
`tben in military hospitals in England until be was
`discbarged from the army witb a disability pension
`and a diagnosis of 'exfoliative dermatitis'.
`In 195R, about a year after onset of tbe eruption,
`he was admitted to Guy's Hospital under Dr L
`Fdrman witb a persisting generalized exudative
`eruption. He had an episode of septicremia and·
`pleurisy at this time whicb responded weil to
`treatment. At tbis time also scrapings from tbe
`skin sbowed fungus which proved on culture to
`be Epidermophyton floccosum. He was given a
`course oforal griseofulvin whicb bad a dramatic
`effect, clearing bis skin in three weeks. He
`remained completely clear for three months after
`whicb tbere was a slight requrrence. From then on
`fungus has always~been·present in tbe finger-nails
`
`Section of Dermatology
`
`745
`
`and toe-nails and occasionally in the groins
`despite repeated courses of griseofulvin.
`There was no further change in bis skin until
`the summer of 1970 wben he developed a
`generalized pruritic eruption. He was admitted to
`St John's Hospital in October 1970, at which time
`he was found to have a low-grade erythroderma
`involving particularly the tnfuk, groins and thighs.
`Several fi.nger- and toe-miils showed minimal
`dystrophy. There were no otbet abnormal physical
`signs. He had furtherbrief admissions in 1971 and
`1972 for investigation. His erythroderma persisted
`more or less unchanged. Treatment with a mixture
`of equal parts ofbenzoic acid compound ointment
`and Propaderm ointment reduced pruritus and
`scaling.
`In June of 1972, during bis last admission, he
`gave a two-month bistory of indigestion, flatu-
`lence and tbe sensation of food sticking in bis
`ehest. A barium meal performed at his local
`hospital in Aberystwyth indicated an ulcer and
`tumour in tbe cardia of the stomach and he was
`referred to the Cardiothoracic Surgical Unit of
`Broadgreen Hospital, Liverpool. <Esopbagoscopy
`was performed and two biopsies taken which
`showed tumour. In September his left ehest and
`abdomen were surgically explored (Mr H R
`Matthews). A tumour of the cardia was found, ·
`partly in the stomach and partly in the cesophagus,
`which was bulky but witb no evidence of spread
`to tbe mediastinal or abdominallymph nodes or .
`liver. <Esophago-gastrectomy Was performed witb
`anastomosis of tbe cesophagus to a gastric tube
`just below the aortic arch. Some local lymph-
`nodes were excised and, in view of the proximity
`of the tumour to the spieen, splenectomy was
`performed as weil. He made a Straightforward
`recovery and has apparently remained weil since.
`The tumour was found on histology to be a
`reticulum cell sarcoma.
`
`lnvestz'gations
`Mycology: E. flo.ccosum bad been found on
`numerous occasions over the years . and,. wa8
`isolated again in toe-nail~, finger-nails and right
`palm in 1970. No f~gus was seen in the scaly red
`lesions on the trunk and shins. The strain of
`E. floccosum isolated from the patient wa:s found
`to be griseofulvin-resistant (minimum inhibitory
`concentration: patient strain >50.00, control
`strain 1.5 p.g/ml; Dr Yvonn~ Clayton). In July
`1972 fungus was found in palins, toes, toe-clefts,
`soles, ballux nails and left finger-nails, but not in
`wrists, trunk, groins, scalp, neck or legs.
`Clinical immunology: Low IgG and IgA Ievels
`were found on tbe four occasions on which
`differential serum immunoglobulins · were esti-
`mated. An average blood IY'mphocytopenia of
`800/mm8 was found with counts below 1000/mma
`on 5 of 7 occa8ions (nornial adult range lOO()J.
`
`Exh. 1035